Delayed rectifier potassium channel subunit

ABSTRACT

The invention provides a human delayed rectifier potassium channel subunit (DRPCS) and polynucleotides which identify and encode DRPCS. The invention also provides expression vectors, host cells, antibodies, agonists, and antagonists. The invention also provides methods for diagnosing, treating or preventing disorders associated with expression of DRPCS.

[0001] This application is a divisional application of copending U.S.application Ser. No. 09/471,468, filed Dec. 22, 1999, which is adivisional application of U.S. Ser. No. 09/069,896, filed Apr. 29, 1998,issued Jun. 6, 2000, as U.S. Pat. No. 6,071,720, entitled “DELAYEDRECTIFIER POTASSIUM CHANNEL SUBUNIT,” both of which are hereby expresslyincorporated by reference.

FIELD OF THE INVENTION

[0002] This invention relates to nucleic acid and amino acid sequencesof a delayed rectifier potassium channel subunit and to the use of thesesequences in the diagnosis, treatment, and prevention of cancer,cardiovascular disorders, and neuronal disorders.

BACKGROUND OF THE INVENTION

[0003] Ion channels are integral membrane proteins, typically comprisedof four subunits, that form highly selective and tightly regulated poresin cellular membranes. Each of these pores controls the influx andefflux of a given ion (e.g., sodium, potassium, calcium, or chloride)across the plasma membrane or the membranes of intracellularcompartments. Essential physiological processes, such as synaptictransmission, stimulation of secretion, fertilization, musclecontraction, and regulation of ion concentrations and pH, depend on thecontrol of ion gradients by ion channels.

[0004] Ion channels open in response to various stimuli. For example,there are ligand gated channels, second-messenger gated channels,voltage gated channels, and shear- or stress-gated channels, as well asleak channels through which ions flow without specific stimulus. Thegating properties characteristic of a given channel include the periodof time it is open (open time), frequency of opening, strength ofstimulus required for activation, and the refractory period. Thesecharacteristics can vary widely based on the subunit composition of thechannel, association with accessory proteins, phosphorylation state, andpost-translational modification.

[0005] Potassium channels are located in all cell types. In neurons andother excitable cells, they set resting membrane potential, regulate keyaspects of the action potential including duration, frequency, andpattern of discharge, and are responsible for repolarization followingan action potential. In non-excitable tissue, potassium channels areinvolved in essential physiological processes including cell proteinsynthesis, control of endocrine secretions, and the maintenance ofosmotic equilibrium across cell membranes and in the plasma. Categoriesof potassium channels include voltage-gated potassium channels,ATP-sensitive potassium channels, second messenger-gated potassiumchannels, and calcium-activated potassium channels.

[0006] Like the voltage-gated channels for sodium and calcium,voltage-gated potassium channels (VGKC) are composed of fourpolypeptides that form homo-oligomers or hetero-oligomers to create thepore through which potassium ions flow. At least ten of thesepotassium-pore-forming subunits, or α subunits, have been described thatfall into four families, designated Kv1-Kv4. Examples of α subunitsinclude the HERG (human ether a go-go) subunit, named after a Drosophilahomolog, and the Kv(LQT)1 subunit. These α subunits share a commonstructural organization which is similar to the α subunits of othervoltage-gated channels. There are six transmembrane-spanning domainswith a short region between the fifth and sixth transmembrane regionsthat senses membrane potential, and the amino and carboxy termini arelocated intracellularly. Current flow through a VGKC can be either anA-type current, which activates at sub-threshold membrane potentials andrapidly inactivates, or a rectifier type current, which activates andinactivates slowly.

[0007] The potassium channel β subunit, also known as the minK proteinor the cardiac delayed rectifier potassium channel protein, is anaccessory subunit that regulates potassium channel gatingcharacteristics, but does not participate in formation of the potassiumpore. At least four variants of this 129 amino acid peptide have beendescribed. They contain a single transmembrane domain, and have beenshown to assemble with at least two different VGKC pore-forming subunitsand regulate their gating characteristics. For example, in mammalianheart, the duration of ventricular action potential is controlled by aninward, rectifying, delayed-type potassium current, which has fast- andslow-activating components. Two α subunits have been shown to mediatethis complex current: the HERG subunit, and the Kv(LQT)1 subunit. Thefast-activating potassium current is mediated by a complex of minK andHERG, while the slow-activating component is mediated by minK andKv(LQT)1. Thus, minK is central to the control of heart rate and rhythm.(Folander, K. et al. (1994) GI 452494; Lai, L. P. et al. (1994) Gene151:339-340; Sanguinetti, M. C. et al. (1996) Nature 384:80-83;McDonald, T. V. et al. (1997) Nature 388:289-292.)

[0008] Potassium channel dysfunctions are associated with a number ofdisease states. For example, potassium channels in smooth muscle tissueof the circulatory system are implicated in hypertension, while thosechannels in the kidney are involved in hypokalemia and the associatedBartter's syndrome and Getelman's syndrome. Both of these syndromes arecharacterized by alterations in potassium metabolism in the kidney.Potassium channels are also involved in certain neuronal disorders.Epileptic seizures can be induced by agents (e.g., pentylenetetrazol)which block potassium channels, most likely due to the loss ofregulation of cellular membrane potentials. A potential role forpotassium channels in Alzheimer's disease has been suggested by studiesdemonstrating that a significant component of senile plaques, betaamyloid or A beta, also blocks voltage-gated potassium channels inhippocampal neurons. (Antes, L. M. et al. (1998) Seminar Nephrol.18:31-45; Stoffel, M. and L. Y. Jan (1998) Nat. Genet. 18:6-8; Madeja,M. et al. (1997) Eur. J. Neurosci. 9:390-395; and Good, T. A. et al.(1996) Biophys. J. 70:296-304.)

[0009] Altered function of minK proteins is involved in long QTsyndrome, a cardiovascular disorder in which patients experience cardiacarrhythmias, fibrillation, syncope, and sudden death. Anothercardiovascular disorder, Jervell and Lange-Nielsen syndrome, is alsoproduced by mutations in minK. Jervell and Lange-Nielsen syndrome ischaracterized by abnormal ventricular repolarization, syncope, andsudden death, and is also associated with congenital deafness. In bothinstances, mutations in the gene encoding a minK homolog are responsiblefor altered gating characteristics of the VGKC, which results indysfunctional potassium channels. (Schulze-Bahr, E. et al. (1997) NatureGenet. 17:267-268; and Splawski, I. et al. (1997) Nature Genet.17:338-340.)

[0010] The discovery of a new delayed rectifier potassium channelsubunit and the polynucleotides encoding it satisfies a need in the artby providing new compositions which are useful in the diagnosis,treatment, and prevention of cancer, cardiovascular disorders, andneuronal disorders.

SUMMARY OF THE INVENTION

[0011] The invention is based on the discovery of a new human delayedrectifier potassium channel subunit (DRPCS), the polynucleotidesencoding DRPCS, and the use of these compositions for the diagnosis,treatment, or prevention of cancer, cardiovascular disorders, andneuronal disorders.

[0012] The invention features a substantially purified polypeptidecomprising the amino acid sequence of SEQ ID NO:1 or a fragment of SEQID NO:1.

[0013] The invention further provides a substantially purified varianthaving at least 90% amino acid sequence identity to the amino acidsequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1. The invention alsoprovides an isolated and purified polynucleotide encoding thepolypeptide comprising the sequence of SEQ ID NO:1 or a fragment of SEQID NO:1. The invention also includes an isolated and purifiedpolynucleotide variant having at least 90% polynucleotide sequenceidentity to the polynucleotide encoding the polypeptide comprising theamino acid sequence of SEQ ID NO: I or a fragment of SEQ ID NO:1.

[0014] The invention further provides an isolated and purifiedpolynucleotide which hybridizes under stringent conditions to thepolynucleotide encoding the polypeptide comprising the amino acidsequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1, as well as anisolated and purified polynucleotide which is complementary to thepolynucleotide encoding the polypeptide comprising the amino acidsequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1.

[0015] The invention also provides an isolated and purifiedpolynucleotide comprising the polynucleotide sequence of SEQ ID NO:2 ora fragment of SEQ ID NO:2, and an isolated and purified polynucleotidevariant having at least 90% polynucleotide sequence identity to thepolynucleotide comprising the polynucleotide sequence of SEQ ID NO:2 ora fragment of SEQ ID NO:2. The invention also provides an isolated andpurified polynucleotide having a sequence complementary to thepolynucleotide comprising the polynucleotide sequence of SEQ ID NO:2 ora fragment of SEQ ID NO:2.

[0016] The invention further provides an expression vector comprising atleast a fragment of the polynucleotide encoding the polypeptidecomprising the sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1. Inanother aspect, the expression vector is contained within a host cell.

[0017] The invention also provides a method for producing a polypeptidecomprising the amino acid sequence of SEQ ID NO:1 or a fragment of SEQID NO:1, the method comprising the steps of: (a) culturing the host cellcomprising an expression vector containing at least a fragment of apolynucleotide encoding the polypeptide comprising the amino acidsequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1 under conditionssuitable for the expression of the polypeptide; and (b) recovering thepolypeptide from the host cell culture.

[0018] The invention also provides a pharmaceutical compositioncomprising a substantially purified polypeptide having the sequence ofSEQ ID NO:1 or a fragment of SEQ ID NO:1 in conjunction with a suitablepharmaceutical carrier.

[0019] The invention further includes a purified antibody which binds toa polypeptide comprising the sequence of SEQ ID NO:1 or a fragment ofSEQ ID NO:1, as well as a purified agonist and a purified antagonist ofthe polypeptide.

[0020] The invention also provides a method for treating or preventing acancer, the method comprising administering to a subject in need of suchtreatment an effective amount of an antagonist of the polypeptide havingthe amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1.

[0021] The invention also provides a method for treating or preventing acardiovascular disorder, the method comprising administering to asubject in need of such treatment an effective amount of apharmaceutical composition comprising substantially purified polypeptidehaving the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ IDNO:1.

[0022] The invention also provides a method for treating or preventing aneuronal disorder, the method comprising administering to a subject inneed of such treatment an effective amount of an antagonist of thepolypeptide having the amino acid sequence of SEQ ID NO:1 or a fragmentof SEQ ID NO:1.

[0023] The invention also provides a method for detecting apolynucleotide encoding a polypeptide comprising the amino acid sequenceof SEQ ID NO:1 or a fragment of SEQ ID NO:1 in a biological samplecontaining nucleic acids, the method comprising the steps of: (a)hybridizing the complement of the polynucleotide encoding thepolypeptide comprising the amino acid sequence of SEQ ID NO:1 or afragment of SEQ ID NO:1 to at least one of the nucleic acids of thebiological sample, thereby forming a hybridization complex; and (b)detecting the hybridization complex, wherein the presence of thehybridization complex correlates with the presence of a polynucleotideencoding the polypeptide comprising the amino acid sequence of SEQ IDNO:1 or a fragment of SEQ ID NO:1 in the biological sample. In oneaspect, the nucleic acids of the biological sample are amplified by thepolymerase chain reaction prior to the hybridizing step.

BRIEF DESCRIPTION OF THE FIGURES

[0024]FIGS. 1A and 1B show the amino acid sequence (SEQ ID NO:1) andnucleic acid sequence (SEQ ID NO:2) of DRPCS. The alignment was producedusing MACDNASIS PRO software (Hitachi Software Engineering Co. Ltd., SanBruno, Calif.).

[0025]FIG. 2 shows the amino acid sequence alignments among DRPCS(Incyte Clone 637471; SEQ ID NO:1), human cardiac delayed rectifierpotassium channel (GI 452494; SEQ ID NO:3), and rat delayed rectifiertype potassium channel (GI 203977; SEQ ID NO:4), produced using themultisequence alignment program of LASERGENE software (DNASTAR Inc,Madison Wis.).

DESCRIPTION OF THE INVENTION

[0026] Before the present proteins, nucleotide sequences, and methodsare described, it is understood that this invention is not limited tothe particular methodology, protocols, cell lines, vectors, and reagentsdescribed, as these may vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to limit the scope of the presentinvention which will be limited only by the appended claims.

[0027] It must be noted that as used herein and in the appended claims,the singular forms “a,” “an,” and “the” include plural reference unlessthe context clearly dictates otherwise. Thus, for example, a referenceto “a host cell” includes a plurality of such host cells, and areference to “an antibody” is a reference to one or more antibodies andequivalents thereof known to those skilled in the art, and so forth.

[0028] Unless defined otherwise, all technical and scientific terms usedherein have the same meanings as commonly understood by one of ordinaryskill in the art to which this invention belongs. Although any methodsand materials similar or equivalent to those described herein can beused in the practice or testing of the present invention, the preferredmethods, devices, and materials are now described. All publicationsmentioned herein are cited for the purpose of describing and disclosingthe cell lines, vectors, and methodologies which are reported in thepublications and which might be used in connection with the invention.Nothing herein is to be construed as an admission that the invention isnot entitled to antedate such disclosure by virtue of prior invention.

DEFINITIONS

[0029] “DRPCS,” as used herein, refers to the amino acid sequences ofsubstantially purified DRPCS obtained from any species, particularly amammalian species, including bovine, ovine, porcine, murine, equine, andpreferably the human species, from any source, whether natural,synthetic, semi-synthetic, or recombinant.

[0030] The term “agonist,” as used herein, refers to a molecule which,when bound to DRPCS, increases or prolongs the duration of the effect ofDRPCS. Agonists may include proteins, nucleic acids, carbohydrates, orany other molecules which bind to and modulate the effect of DRPCS.

[0031] An “allelic variant,” as this term is used herein, is analternative form of the gene encoding DRPCS. Allelic variants may resultfrom at least one mutation in the nucleic acid sequence and may resultin altered mRNAs or in polypeptides whose structure or function may ormay not be altered. Any given natural or recombinant gene may have none,one, or many allelic forms. Common mutational changes which give rise toallelic variants are generally ascribed to natural deletions, additions,or substitutions of nucleotides. Each of these types of changes mayoccur alone, or in combination with the others, one or more times in agiven sequence.

[0032] “Altered” nucleic acid sequences encoding DRPCS, as describedherein, include those sequences with deletions, insertions, orsubstitutions of different nucleotides, resulting in a polynucleotidethe same as DRPCS or a polypeptide with at least one functionalcharacteristic of DRPCS. Included within this definition arepolymorphisms which may or may not be readily detectable using aparticular oligonucleotide probe of the polynucleotide encoding DRPCS,and improper or unexpected hybridization to allelic variants, with alocus other than the normal chromosomal locus for the polynucleotidesequence encoding DRPCS. The encoded protein may also be “altered,” andmay contain deletions, insertions, or substitutions of amino acidresidues which produce a silent change and result in a functionallyequivalent DRPCS. Deliberate amino acid substitutions may be made on thebasis of similarity in polarity, charge, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the residues, as longas the biological or immunological activity of DRPCS is retained. Forexample, negatively charged amino acids may include aspartic acid andglutamic acid, positively charged amino acids may include lysine andarginine, and amino acids with uncharged polar head groups havingsimilar hydrophilicity values may include leucine, isoleucine, andvaline; glycine and alanine; asparagine and glutamine; serine andthreonine; and phenylalanine and tyrosine.

[0033] The terms “amino acid” or “amino acid sequence,” as used herein,refer to an oligopeptide, peptide, polypeptide, or protein sequence, ora fragment of any of these, and to naturally occurring or syntheticmolecules. In this context, “fragments,” “immunogenic fragments,” or“antigenic fragments” refer to fragments of DRPCS which are preferablyabout 5 to about 15 amino acids in length, most preferably 14 aminoacids, and which retain some biological activity or immunologicalactivity of DRPCS. Where “amino acid sequence” is recited herein torefer to an amino acid sequence of a naturally occurring proteinmolecule, “amino acid sequence” and like terms are not meant to limitthe amino acid sequence to the complete native amino acid sequenceassociated with the recited protein molecule.

[0034] “Amplification,” as used herein, relates to the production ofadditional copies of a nucleic acid sequence. Amplification is generallycarried out using polymerase chain reaction (PCR) technologies wellknown in the art. (See, e.g., Dieffenbach, C. W. and G. S. Dveksler(1995) PCR Primer, a Laboratory Manual, Cold Spring Harbor Press,Plainview, N.Y., pp. 1-5.)

[0035] The term “antagonist,” as it is used herein, refers to a moleculewhich, when bound to DRPCS, decreases the amount or the duration of theeffect of the biological or immunological activity of DRPCS. Antagonistsmay include proteins, nucleic acids, carbohydrates, antibodies, or anyother molecules which decrease the effect of DRPCS.

[0036] As used herein, the term “antibody” refers to intact molecules aswell as to fragments thereof, such as Fab, F(ab′)₂, and Fv fragments,which are capable of binding the epitopic determinant. Antibodies thatbind DRPCS polypeptides can be prepared using intact polypeptides orusing fragments containing small peptides of interest as the immunizingantigen. The polypeptide or oligopeptide used to immunize an animal(e.g., a mouse, a rat, or a rabbit) can be derived from the translationof RNA, or synthesized chemically, and can be conjugated to a carrierprotein if desired. Commonly used carriers that are chemically coupledto peptides include bovine serum albumin, thyroglobulin, and keyholelimpet hemocyanin (KLH). The coupled peptide is then used to immunizethe animal.

[0037] The term “antigenic determinant,” as used herein, refers to thatfragment of a molecule (i.e., an epitope) that makes contact with aparticular antibody. When a protein or a fragment of a protein is usedto immunize a host animal, numerous regions of the protein may inducethe production of antibodies which bind specifically to antigenicdeterminants (given regions or three-dimensional structures on theprotein). An antigenic determinant may compete with the intact antigen(i.e., the immunogen used to elicit the immune response) for binding toan antibody.

[0038] The term “antisense,” as used herein, refers to any compositioncontaining a nucleic acid sequence which is complementary to the “sense”strand of a specific nucleic acid sequence. Antisense molecules may beproduced by any method including synthesis or transcription. Onceintroduced into a cell, the complementary nucleotides combine withnatural sequences produced by the cell to form duplexes and to blockeither transcription or translation. The designation “negative” canrefer to the antisense strand, and the designation “positive” can referto the sense strand.

[0039] As used herein, the term “biologically active” refers to aprotein having structural, regulatory, or biochemical functions of anaturally occurring molecule. Likewise, “immunologically active” refersto the capability of the natural, recombinant, or synthetic DRPCS, or ofany oligopeptide thereof, to induce a specific immune response inappropriate animals or cells and to bind with specific antibodies.

[0040] The terms “complementary” or “complementarity,” as used herein,refer to the natural binding of polynucleotides under permissive saltand temperature conditions by base pairing. For example, the sequence“A-G-T” binds to the complementary sequence “T-C-A.” Complementaritybetween two single-stranded molecules may be “partial,” such that onlysome of the nucleic acids bind, or it may be “complete,” such that totalcomplementarity exists between the single stranded molecules. The degreeof complementarity between nucleic acid strands has significant effectson the efficiency and strength of the hybridization between the nucleicacid strands. This is of particular importance in amplificationreactions, which depend upon binding between nucleic acids strands, andin the design and use of peptide nucleic acid (PNA) molecules.

[0041] A “composition comprising a given polynucleotide sequence” or a“composition comprising a given amino acid sequence,” as these terms areused herein, refer broadly to any composition containing the givenpolynucleotide or amino acid sequence. The composition may comprise adry formulation, an aqueous solution, or a sterile composition.Compositions comprising polynucleotide sequences encoding DRPCS orfragments of DRPCS may be employed as hybridization probes. The probesmay be stored in freeze-dried form and may be associated with astabilizing agent such as a carbohydrate. In hybridizations, the probemay be deployed in an aqueous solution containing salts, e.g., NaCl,detergents, e.g.,sodium dodecyl sulfate (SDS), and other components,e.g., Denhardt's solution, dry milk, salmon sperm DNA, etc.

[0042] “Consensus sequence,” as used herein, refers to a nucleic acidsequence which has been resequenced to resolve uncalled bases, extendedusing the XL-PCR kit (Perkin Elmer, Norwalk, Conn,.) in the 5′ and/orthe 3′ direction, and resequenced, or which has been assembled from theoverlapping sequences of more than one Incyte Clone using a computerprogram for fragment assembly, such as the GELVIEW fragment assemblysystem (GCG, Madison, Wis.). Some sequences have been both extended andassembled to produce the consensus sequence.

[0043] As used herein, the term “correlates with expression of apolynucleotide” indicates that the detection of the presence of nucleicacids, the same or related to a nucleic acid sequence encoding DRPCS, byNorthern analysis is indicative of the presence of nucleic acidsencoding DRPCS in a sample, and thereby correlates with expression ofthe transcript from the polynucleotide encoding DRPCS.

[0044] A “deletion,” as the term is used herein, refers to a change inthe amino acid or nucleotide sequence that results in the absence of oneor more amino acid residues or nucleotides.

[0045] The term “derivative,” as used herein, refers to the chemicalmodification of a polypeptide sequence, or a polynucleotide sequence.Chemical modifications of a polynucleotide sequence can include, forexample, replacement of hydrogen by an alkyl, acyl, or amino group. Aderivative polynucleotide encodes a polypeptide which retains at leastone biological or immunological function of the natural molecule. Aderivative polypeptide is one modified by glycosylation, pegylation, orany similar process that retains at least one biological orimmunological function of the polypeptide from which it was derived.

[0046] The term “similarity,” as used herein, refers to a degree ofcomplementarity. There may be partial similarity or complete similarity.The word “identity” may substitute for the word “similarity.” Apartially complementary sequence that at least partially inhibits anidentical sequence from hybridizing to a target nucleic acid is referredto as “substantially similar.” The inhibition of hybridization of thecompletely complementary sequence to the target sequence may be examinedusing a hybridization assay (Southern or Northern blot, solutionhybridization, and the like) under conditions of reduced stringency. Asubstantially similar sequence or hybridization probe will compete forand inhibit the binding of a completely similar (identical) sequence tothe target sequence under conditions of reduced stringency. This is notto say that conditions of reduced stringency are such that non-specificbinding is permitted, as reduced stringency conditions require that thebinding of two sequences to one another be a specific (i.e., aselective) interaction. The absence of non-specific binding may betested by the use of a second target sequence which lacks even a partialdegree of complementarity (e.g., less than about 30% similarity oridentity). In the absence of non-specific binding, the substantiallysimilar sequence or probe will not hybridize to the secondnon-complementary target sequence.

[0047] The phrases “percent identity” or “% identity” refer to thepercentage of sequence similarity found in a comparison of two or moreamino acid or nucleic acid sequences. Percent identity can be determinedelectronically, e.g., by using the MEGALIGN program (DNASTAR, Inc.,Madison Wis.). The MEGALIGN program can create alignments between two ormore sequences according to different methods, e.g., the clustal method.(See, e.g., Higgins, D. G. and P. M. Sharp (1988) Gene 73:237-244.) Theclustal algorithm groups sequences into clusters by examining thedistances between all pairs. The clusters are aligned pairwise and thenin groups. The percentage similarity between two amino acid sequences,e.g., sequence A and sequence B, is calculated by dividing the length ofsequence A, minus the number of gap residues in sequence A, minus thenumber of gap residues in sequence B, into the sum of the residuematches between sequence A and sequence B, times one hundred. Gaps oflow or of no similarity between the two amino acid sequences are notincluded in determining percentage similarity. Percent identity betweennucleic acid sequences can also be counted or calculated by othermethods known in the art, e.g., the Jotun Hein method. (See, e.g., Hein,J. (1990) Methods Enzymol. 183:626-645.) Identity between sequences canalso be determined by other methods known in the art, e.g., by varyinghybridization conditions.

[0048] “Human artificial chromosomes” (HACs), as described herein, arelinear microchromosomes which may contain DNA sequences of about 6 kb to10 Mb in size, and which contain all of the elements required for stablemitotic chromosome segregation and maintenance. (See, e.g., Harrington,J. J. et al. (1997) Nat. Genet. 15:345-355.)

[0049] The term “humanized antibody,” as used herein, refers to antibodymolecules in which the amino acid sequence in the non-antigen bindingregions has been altered so that the antibody more closely resembles ahuman antibody, and still retains its original binding ability.

[0050] “Hybridization,” as the term is used herein, refers to anyprocess by which a strand of to nucleic acid binds with a complementarystrand through base pairing.

[0051] As used herein, the term “hybridization complex” refers to acomplex formed between two nucleic acid sequences by virtue of theformation of hydrogen bonds between complementary bases. A hybridizationcomplex may be formed in solution (e.g., Cot or Rot analysis) or formedbetween one nucleic acid sequence present in solution and anothernucleic acid sequence immobilized on a solid support (e.g., paper,membranes, filters, chips, pins or glass slides, or any otherappropriate substrate to which cells or their nucleic acids have beenfixed).

[0052] The words “insertion” or “addition,” as used herein, refer tochanges in an amino acid or nucleotide sequence resulting in theaddition of one or more amino acid residues or nucleotides,respectively, to the sequence found in the naturally occurring molecule.

[0053] “Immune response” can refer to conditions associated withinflammation, trauma, immune disorders, or infectious or geneticdisease, etc. These conditions can be characterized by expression ofvarious factors, e.g., cytokines, chemokines, and other signalingmolecules, which may affect cellular and systemic defense systems.

[0054] The term “microarray,” as used herein, refers to an arrangementof distinct polynucleotides arrayed on a substrate, e.g., paper, nylonor any other type of membrane, filter, chip, glass slide, or any othersuitable solid support.

[0055] The terms “element” or “array element” as used herein in amicroarray context, refer to hybridizable polynucleotides arranged onthe surface of a substrate.

[0056] The term “modulate,” as it appears herein, refers to a change inthe activity of DRPCS. For example, modulation may cause an increase ora decrease in protein activity, binding characteristics, or any otherbiological, functional, or immunological properties of DRPCS.

[0057] The phrases “nucleic acid” or “nucleic acid sequence,” as usedherein, refer to a nucleotide, oligonucleotide, polynucleotide, or anyfragment thereof. These phrases also refer to DNA or RNA of genomic orsynthetic origin which may be single-stranded or double-stranded and mayrepresent the sense or the antisense strand, to peptide nucleic acid(PNA), or to any DNA-like or RNA-like material. In this context,“fragments” refers to those nucleic acid sequences which, whentranslated, would produce polypeptides retaining some functionalcharacteristic, e.g., antigenicity, or structural domain characteristic,e.g., ATP-binding site, of the full-length polypeptide.

[0058] The terms “operably associated” or “operably linked,” as usedherein, refer to functionally related nucleic acid sequences. A promoteris operably associated or operably linked with a coding sequence if thepromoter controls the translation of the encoded polypeptide. Whileoperably associated or operably linked nucleic acid sequences can becontiguous and in the same reading frame, certain genetic elements,e.g., repressor genes, are not contiguously linked to the sequenceencoding the polypeptide but still bind to operator sequences thatcontrol expression of the polypeptide.

[0059] The term “oligonucleotide,” as used herein, refers to a nucleicacid sequence of at least about 6 nucleotides to 60 nucleotides,preferably about 15 to 30 nucleotides, and most preferably about 20 to25 nucleotides, which can be used in PCR amplification or in ahybridization assay or microarray. As used herein, the term“oligonucleotide” is substantially equivalent to the terms “amplimer,”“primer,” “oligomer,” and “probe,” as these terms are commonly definedin the art.

[0060] “Peptide nucleic acid” (PNA), as used herein, refers to anantisense molecule or anti-gene agent which comprises an oligonucleotideof at least about 5 nucleotides in length linked to a peptide backboneof amino acid residues ending in lysine. The terminal lysine conferssolubility to the composition. PNAs preferentially bind complementarysingle stranded DNA or RNA and stop transcript elongation, and may bepegylated to extend their lifespan in the cell. (See, e.g., Nielsen, P.E. et al. (1993) Anticancer Drug Des. 8:53-63.)

[0061] The term “sample,” as used herein, is used in its broadest sense.A biological sample suspected of containing nucleic acids encodingDRPCS, or fragments thereof, or DRPCS itself, may comprise a bodilyfluid; an extract from a cell, chromosome, organelle, or membraneisolated from a cell; a cell; genomic DNA, RNA, or cDNA, in solution orbound to a solid support; a tissue; a tissue print; etc.

[0062] As used herein, the terms “specific binding” or “specificallybinding” refer to that interaction between a protein or peptide and anagonist, an antibody, or an antagonist. The interaction is dependentupon the presence of a particular structure of the protein, e.g., theantigenic determinant or epitope, recognized by the binding molecule.For example, if an antibody is specific for epitope “A,” the presence ofa polypeptide containing the epitope A, or the presence of freeunlabeled A, in a reaction containing free labeled A and the antibodywill reduce the amount of labeled A that binds to the antibody.

[0063] As used herein, the term “stringent conditions” refers toconditions which permit hybridization between polynucleotides and theclaimed polynucleotides. Stringent conditions can be defined by saltconcentration, the concentration of organic solvent (e.g., formamide),temperature, and other conditions well known in the art. In particular,stringency can be increased by reducing the concentration of salt,increasing the concentration of formamide, or raising the hybridizationtemperature.

[0064] For example, stringent salt concentration will ordinarily be lessthan about 750 mM NaCl and 75 mM trisodium citrate, preferably less thanabout 500 mM NaCl and 50 mM trisodium citrate, and most preferably lessthan about 250 mM NaCl and 25 mM trisodium citrate. Low stringencyhybridization can be obtained in the absence of organic solvent, e.g.,formamide, while high stringency hybridization can be obtained in thepresence of at least about 35% formamide, and most preferably at leastabout 50% formamide. Stringent temperature conditions will ordinarilyinclude temperatures of at least about 30° C., more preferably of atleast about 37° C., and most preferably of at least about 42° C. Varyingadditional parameters, such as hybridization time, the concentration ofdetergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion orexclusion of carrier DNA, are well known to those skilled in the art.Various levels of stringency are accomplished by combining these variousconditions as needed. In a preferred embodiment, hybridization willoccur at 30° C. in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. Ina more preferred embodiment, hybridization will occur at 37° C. in 500mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 kg/mldenatured salmon sperm DNA (ssDNA). In a most preferred embodiment,hybridization will occur at 42° C. in 250 mM NaCl, 25 mM trisodiumcitrate, 1% SDS, 50% formamide, and 200 μg/ml ssDNA. Useful variationson these conditions will be readily apparent to those skilled in theart.

[0065] The washing steps which follow hybridization can also vary instringency. Wash stringency conditions can be defined by saltconcentration and by temperature. As above, wash stringency can beincreased by decreasing salt concentration or by increasing temperature.For example, stringent salt concentration for the wash steps willpreferably be less than about 30 mM NaCl and 3 mM trisodium citrate, andmost preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate.Stringent temperature conditions for the wash steps will ordinarilyinclude temperatures of at least about 25° C., more preferably of atleast about 42° C., and most preferably of at least about 68° C. In apreferred embodiment, wash steps will occur at 25° C. in 30 mM NaCl, 3mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, washsteps will occur at 42° C. in 15 mM NaCl, 1.5 mM trisodium citrate, and0.1% SDS. In a most preferred embodiment, wash steps will occur at 68°C. in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additionalvariations on these conditions will be readily apparent to those skilledin the art.

[0066] The term “substantially purified,” as used herein, refers tonucleic acid or amino acid sequences that are removed from their naturalenvironment and are isolated or separated, and are at least about 60%free, preferably about 75% free, and most preferably about 90% free fromother components with which they are naturally associated.

[0067] A “substitution,” as used herein, refers to the replacement ofone or more amino acids or nucleotides by different amino acids ornucleotides, respectively.

[0068] “Transformation,” as defined herein, describes a process by whichexogenous DNA enters and changes a recipient cell. Transformation mayoccur under natural or artificial conditions according to variousmethods well known in the art, and may rely on any known method for theinsertion of foreign nucleic acid sequences into a prokaryotic oreukaryotic host cell. The method for transformation is selected based onthe type of host cell being transformed and may include, but is notlimited to, viral infection, electroporation, heat shock, lipofection,and particle bombardment. The term “transformed” cells includes stablytransformed cells in which the inserted DNA is capable of replicationeither as an autonomously replicating plasmid or as part of the hostchromosome, as well as transiently transformed cells which express theinserted DNA or RNA for limited periods of time.

[0069] A “variant” of DRPCS, as used herein, refers to an amino acidsequence that is altered by one or more amino acids. The variant mayhave “conservative” changes, wherein a substituted amino acid hassimilar structural or chemical properties (e.g., replacement of leucinewith isoleucine). More rarely, a variant may have “nonconservative”changes (e.g., replacement of glycine with tryptophan). Analogous minorvariations may also include amino acid deletions or insertions, or both.Guidance in determining which amino acid residues may be substituted,inserted, or deleted without abolishing biological or immunologicalactivity may be found using computer programs well known in the art, forexample, LASERGENE software (DNASTAR Inc).

THE INVENTION

[0070] The invention is based on the discovery of a new human delayedrectifier potassium channel subunit (DRPCS), the polynucleotidesencoding DRPCS, and the use of these compositions for the diagnosis,treatment, or prevention of cancer, cardiovascular disorders, andneuronal disorders.

[0071] Nucleic acids encoding the DRPCS of the present invention werefirst identified in Incyte Clone 637471 from the breast tissue cDNAlibrary (BRSTNOT03) using a computer search, e.g., BLAST, for amino acidsequence alignments. A consensus sequence, SEQ ID NO:2, was derived fromthis clone.

[0072] In one embodiment, the invention encompasses a polypeptidecomprising the amino acid sequence of SEQ ID NO:1, as shown in FIGS. 1Aand 1B. DRPCS is 170 amino acids in length and has a predictedtransmembrane region from about residue F₃₇ through L₅₅, and twopotential N-glycosylation sites at residues N₈ and N₁₅₆. In addition,DRPCS has a potential cAMP- and cGMP-dependent protein kinasephosphorylation site at residue S₆₈, three potential casein kinase IIphosphorylation sites at residues S₁₉, S₁₃₀, and T₁₃₇, and a potentialprotein kinase C phosphorylation site at residue S₆₀, as well as apotential glycosaminoglycan attachment site at S₃₀-PRINTS analysisidentifies DRPCS as a potassium channel (PR00168), which the algorithmdefines using four regions designated PR00168A, PR00168B, PR00168C andPR00168D. The region from Y₃₈ through G₅₂, matching region PR00168C,received a score of 1282 on a strength of 1262, and was supported by thepresence of region PR00168D with a P value less than 3.1×10⁻⁵. As shownin FIG. 2, DRPCS has chemical and structural similarity with delayedrectifier potassium channels from human (hDRPC) (GI 452494; SEQ ID NO:3)and rat (rDRPC) (GI 203977). In particular, DRPCS and hDRPC share 20%identity, while DRPCS and rDRPC share 21% identity. In the predictedtransmembrane domain, DRPCS has a high level of similarity with bothhDRPC and rDRPC (FIG. 2). Northern analysis shows the expression of thissequence in various libraries, at least 50% of which are immortalized orcancerous. Of particular note is the expression of DRPCS in librariesfrom cardiovascular and reproductive tissues. The fragment of SEQ IDNO:2 from about nucleotide 93 through about nucleotide 153 is useful,e.g., as a hybridization probe.

[0073] The invention also encompasses DRPCS variants. A preferred DRPCSvariant is one which has at least about 80%, more preferably at leastabout 90%, and most preferably at least about 95% amino acid sequenceidentity to the DRPCS amino acid sequence, and which contains at leastone functional or structural characteristic of DRPCS.

[0074] The invention also encompasses polynucleotides which encodeDRPCS. In a particular embodiment, the invention encompasses apolynucleotide sequence comprising the sequence of SEQ ID NO:2, whichencodes a DRPCS.

[0075] The invention also encompasses a variant of a polynucleotidesequence encoding DRPCS. In particular, such a variant polynucleotidesequence will have at least about 80%, more preferably at least about90%, and most preferably at least about 95% polynucleotide sequenceidentity to the polynucleotide sequence encoding DRPCS. A particularaspect of the invention encompasses a variant of SEQ ID NO:2 which hasat least about 80%, more preferably at least about 90%, and mostpreferably at least about 95% polynucleotide sequence identity to SEQ IDNO:2. Any one of the polynucleotide variants described above can encodean amino acid sequence which contains at least one functional orstructural characteristic of DRPCS.

[0076] It will be appreciated by those skilled in the art that as aresult of the degeneracy of the genetic code, a multitude ofpolynucleotide sequences encoding DRPCS, some bearing minimal similarityto the polynucleotide sequences of any known and naturally occurringgene, may be produced. Thus, the invention contemplates each and everypossible variation of polynucleotide sequence that could be made byselecting combinations based on possible codon choices. Thesecombinations are made in accordance with the standard triplet geneticcode as applied to the polynucleotide sequence of naturally occurringDRPCS, and all such variations are to be considered as beingspecifically disclosed.

[0077] Although nucleotide sequences which encode DRPCS and its variantsare preferably capable of hybridizing to the nucleotide sequence of thenaturally occurring DRPCS under appropriately selected conditions ofstringency, it may be advantageous to produce nucleotide sequencesencoding DRPCS or its derivatives possessing a substantially differentcodon usage, e.g., inclusion of non-naturally occurring codons. Codonsmay be selected to increase the rate at which expression of the peptideoccurs in a particular prokaryotic or eukaryotic host in accordance withthe frequency with which particular codons are utilized by the host.Other reasons for substantially altering the nucleotide sequenceencoding DRPCS and its derivatives without altering the encoded aminoacid sequences include the production of RNA transcripts having moredesirable properties, such as a greater half-life, than transcriptsproduced from the naturally occurring sequence.

[0078] The invention also encompasses production of DNA sequences whichencode DRPCS and DRPCS derivatives, or fragments thereof, entirely bysynthetic chemistry. After production, the synthetic sequence may beinserted into any of the many available expression vectors and cellsystems using reagents well known in the art. Moreover, syntheticchemistry may be used to introduce mutations into a sequence encodingDRPCS or any fragment thereof.

[0079] Also encompassed by the invention are polynucleotide sequencesthat are capable of hybridizing to the claimed polynucleotide sequences,and, in particular, to those shown in SEQ ID NO:2, or a fragment of SEQID NO:2, under various conditions of stringency. (See, e.g., Wahl, G. M.and S. L. Berger (1987) Methods Enzymol. 152:399-407; Kimmel, A. R.(1987) Methods Enzymol. 152:507-511.)

[0080] Methods for DNA sequencing are well known and generally availablein the art and may be used to practice any of the embodiments of theinvention. The methods may employ such enzymes as the Klenow fragment ofDNA polymerase I, SEQUENASE (US Biochemical Corp., Cleveland, Ohio), Taqpolymerase (Perkin Elmer), thermostable T7 polymerase (Amersham,Chicago, Ill.), or combinations of polymerases and proofreadingexonucleases such as those found in the ELONGASE amplification system(GIBCO BRL, Gaithersburg, Md.). Preferably, the process is automatedwith machines such as the MICROLAB 2200 liquid transfer system(Hamilton, Reno, Nev.), PTC200 thermal cycler (MJ Research, Watertown,Mass.) and the ABI CATALYST and 373 and 377 DNA sequencers (PerkinElmer).

[0081] The nucleic acid sequences encoding DRPCS may be extendedutilizing a partial nucleotide sequence and employing various PCR-basedmethods known in the art to detect upstream sequences, such as promotersand regulatory elements. For example, one method which may be employed,restriction-site PCR, uses universal and nested primers to amplifyunknown sequence from genomic DNA within a cloning vector. (See, e.g.,Sarkar, G. (1993) PCR Methods Applic. 2:318-322.) Another method,inverse PCR, uses primers that extend in divergent directions to amplifyunknown sequence from a circularized template. The template is derivedfrom restriction fragments comprising a known genomic locus andsurrounding sequences. (See, e.g., Triglia, T. et al. (1988) NucleicAcids Res. 16:8186.) A third method, capture PCR, involves PCRamplification of DNA fragments adjacent to known sequences in human andyeast artificial chromosome DNA. (See, e.g., Lagerstrom, M. et al.(1991) PCR Methods Applic. 1:111-119.) In this method, multiplerestriction enzyme digestions and ligations may be used to insert anengineered double-stranded sequence into a region of unknown sequencebefore performing PCR. Other methods which may be used to retrieveunknown sequences are known in the art. (See, e.g., Parker, J. D. et al.(1991) Nucleic Acids Res. 19:3055-3060). Additionally, one may use PCR,nested primers, and PROMOTERFINDER libraries to walk genomic DNA(Clontech, Palo Alto, Calif.). This procedure avoids the need to screenlibraries and is useful in finding intron/exon junctions. For allPCR-based methods, primers may be designed using commercially availablesoftware, such as OLIGO 4.06 primer analysis software (NationalBiosciences Inc., Plymouth, Minn.) or another appropriate program, to beabout 22 to 30 nucleotides in length, to have a GC content of about 50%or more, and to anneal to the template at temperatures of about 68° C.to 72° C.

[0082] When screening for full-length cDNAs, it is preferable to uselibraries that have been size-selected to include larger cDNAs. Inaddition, random-primed libraries, which often include sequencescontaining the 5′ regions of genes, are preferable for situations inwhich an oligo d(T) library does not yield a full-length cDNA. Genomiclibraries may be useful for extension of sequence into 5′non-transcribed regulatory regions.

[0083] Capillary electrophoresis systems which are commerciallyavailable may be used to analyze the size or confirm the nucleotidesequence of sequencing or PCR products. In particular, capillarysequencing may employ flowable polymers for electrophoretic separation,four different nucleotide-specific, laser-stimulated fluorescent dyes,and a charge coupled device camera for detection of the emittedwavelengths. Output/light intensity may be converted to electricalsignal using appropriate software (e.g., GENOTYPER and SEQUENCENAVIGATOR, Perkin Elmer), and the entire process from loading of samplesto computer analysis and electronic data display may be computercontrolled. Capillary electrophoresis is especially preferable forsequencing small DNA fragments which may be present in limited amountsin a particular sample.

[0084] In another embodiment of the invention, polynucleotide sequencesor fragments thereof which encode DRPCS may be cloned in recombinant DNAmolecules that direct expression of DRPCS, or fragments or functionalequivalents thereof, in appropriate host cells. Due to the inherentdegeneracy of the genetic code, other DNA sequences which encodesubstantially the same or a functionally equivalent amino acid sequencemay be produced and used to express DRPCS.

[0085] The nucleotide sequences of the present invention can beengineered using methods generally known in the art in order to alterDRPCS-encoding sequences for a variety of purposes including, but notlimited to, modification of the cloning, processing, and/or expressionof the gene product. DNA shuffling by random fragmentation and PCRreassembly of gene fragments and synthetic oligonucleotides may be usedto engineer the nucleotide sequences. For example,oligonucleotide-mediated site-directed mutagenesis may be used tointroduce mutations that create new restriction sites, alterglycosylation patterns, change codon preference, produce splicevariants, and so forth.

[0086] In another embodiment, sequences encoding DRPCS may besynthesized, in whole or in part, using chemical methods well known inthe art. (See, e.g., Caruthers, M. H. et al. (1980) Nucl. Acids Res.Symp. Ser. 215-223, and Horn, T. et al. (1980) Nucl. Acids Res. Symp.Ser. 225-232.) Alternatively, DRPCS itself or a fragment thereof may besynthesized using chemical methods. For example, peptide synthesis canbe performed using various solid-phase techniques. (See, e.g., Roberge,J. Y. et al. (1995) Science 269:202-204.) Automated synthesis may beachieved using the ABI 431A peptide synthesizer (Perkin Elmer).Additionally, the amino acid sequence of DRPCS, or any part thereof, maybe altered during direct synthesis and/or combined with sequences fromother proteins, or any part thereof, to produce a variant polypeptide.

[0087] The peptide may be substantially purified by preparative highperformance liquid chromatography. (See, e.g, Chiez, R. M. and F. Z.Regnier (1990) Methods Enzymol. 182:392-421.) The composition of thesynthetic peptides may be confirmed by amino acid analysis or bysequencing. (See, e.g., Creighton, T. (1984) Proteins, Structures andMolecular Properties, W H Freeman and Co., New York, N.Y.)

[0088] In order to express a biologically active DRPCS, the nucleotidesequences encoding DRPCS or derivatives thereof may be inserted into anappropriate expression vector, i.e., a vector which contains thenecessary elements for transcriptional and translational control of theinserted coding sequence in a suitable host. These elements includeregulatory sequences, such as enhancers, constitutive and induciblepromoters, and 5′ and 3′ untranslated regions in the vector and inpolynucleotide sequences encoding DRPCS. Such elements may vary in theirstrength and specificity. Specific initiation signals may also be usedto achieve more efficient translation of sequences encoding DRPCS. Suchsignals include the ATG initiation codon and adjacent sequences, e.g.the Kozak sequence. In cases where sequences encoding DRPCS and itsinitiation codon and upstream regulatory sequences are inserted into theappropriate expression vector, no additional transcriptional ortranslational control signals may be needed. However, in cases whereonly coding sequence, or a fragment thereof, is inserted, exogenoustranslational control signals including an in-frame ATG initiation codonshould be provided by the vector. Exogenous translational elements andinitiation codons may be of various origins, both natural and synthetic.The efficiency of expression may be enhanced by the inclusion ofenhancers appropriate for the particular host cell system used. (See,e.g., Scharf, D. et al. (1994) Results Probl. Cell Differ. 20:125-162.)

[0089] Methods which are well known to those skilled in the art may beused to construct expression vectors containing sequences encoding DRPCSand appropriate transcriptional and translational control elements.These methods include in vitro recombinant DNA techniques, synthetictechniques, and in vivo genetic recombination. (See, e.g., Sambrook, J.et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring HarborPress, Plainview, N.Y., ch. 4, 8, and 16-17; and Ausubel, F. M. et al.(1995, and periodic supplements) Current Protocols in Molecular Biology,John Wiley & Sons, New York, N.Y., ch. 9, 13, and 16.)

[0090] A variety of expression vector/host systems may be utilized tocontain and express sequences encoding DRPCS. These include, but are notlimited to, microorganisms such as bacteria transformed with recombinantbacteriophage, plasmid, or cosmid DNA expression vectors; yeasttransformed with yeast expression vectors; insect cell systems infectedwith viral expression vectors (e.g., baculovirus); plant cell systemstransformed with viral expression vectors (e.g., cauliflower mosaicvirus (CaMV) or tobacco mosaic virus (TMV)) or with bacterial expressionvectors (e.g., Ti or pBR322 plasmids); or animal cell systems. Theinvention is not limited by the host cell employed.

[0091] In bacterial systems, a number of cloning and expression vectorsmay be selected depending upon the use intended for polynucleotidesequences encoding DRPCS. For example, routine cloning, subcloning, andpropagation of polynucleotide sequences encoding DRPCS can be achievedusing a multifunctional E. coli vector such as PBLUESCRIPT (Stratagene)or PSPORT1 plasmid (GIBCO BRL). Ligation of sequences encoding DRPCSinto the vector's multiple cloning site disrupts the lacZ gene, allowinga colorimetric screening procedure for identification of transformedbacteria containing recombinant molecules. In addition, these vectorsmay be useful for in vitro transcription, dideoxy sequencing, singlestrand rescue with helper phage, and creation of nested deletions in thecloned sequence. (See, e.g., Van Heeke, G. and S. M. Schuster (1989) J.Biol. Chem. 264:5503-5509.) When large quantities of DRPCS are needed,e.g. for the production of antibodies, vectors which direct high levelexpression of DRPCS may be used. For example, vectors containing thestrong, inducible T5 or T7 bacteriophage promoter may be used.

[0092] Yeast expression systems may be used for production of DRPCS. Anumber of vectors containing constitutive or inducible promoters, suchas alpha factor, alcohol oxidase, and PGH, may be used in the yeastSaccharomyces cerevisiae or Pichia pastoris. In addition, such vectorsdirect either the secretion or intracellular retention of expressedproteins and enable integration of foreign sequences into the hostgenome for stable propagation. (See, e.g., Ausubel, supra; Bitter, G. A.et al. (1987) Methods Enzymol. 153:516-544; and Scorer, C. A. et al.(1994) Bio/Technology 12:181-184.)

[0093] Plant systems may also be used for expression of DRPCS.Transcription of sequences encoding DRPCS may be driven viral promoters,e.g., the 35S and 19S promoters of CaMV used alone or in combinationwith the omega leader sequence from TMV. (Takamatsu, N. (1987) EMBO J.6:307-311.) Alternatively, plant promoters such as the small subunit ofRUBISCO or heat shock promoters may be used. (See, e.g., Coruzzi, G. etal. (1984) EMBO J. 3:1671-1680; Broglie, R. et al. (1984) Science224:838-843; and Winter, J. et al. (1991) Results Probl. Cell Differ.17:85-105.) These constructs can be introduced into plant cells bydirect DNA transformation or pathogen-mediated transfection. (See, e.g.,Hobbs, S. or Murry, L. E. in McGraw Hill Yearbook of Science andTechnology (1992) McGraw Hill, New York, N.Y.; pp. 191-196.)

[0094] In mammalian cells, a number of viral-based expression systemsmay be utilized. In cases where an adenovirus is used as an expressionvector, sequences encoding DRPCS may be ligated into an adenovirustranscription/translation complex consisting of the late promoter andtripartite leader sequence. Insertion in a non-essential E1 or E3 regionof the viral genome may be used to obtain infective virus whichexpresses DRPCS in host cells. (See, e.g., Logan, J. and T. Shenk (1984)Proc. Natl. Acad. Sci. 81:3655-3659.) In addition, transcriptionenhancers, such as the Rous sarcoma virus (RSV) enhancer, may be used toincrease expression in mammalian host cells. SV40 or EBV-based vectorsmay also be used for high-level protein expression.

[0095] Human artificial chromosomes (HACs) may also be employed todeliver larger fragments of DNA than can be contained in and expressedfrom a plasmid. HACs of about 6 kb to 10 Mb are constructed anddelivered via conventional delivery methods (liposomes, polycationicamino polymers, or vesicles) for therapeutic purposes.

[0096] For long term production of recombinant proteins in mammaliansystems, stable expression of DRPCS in cell lines is preferred. Forexample, sequences encoding DRPCS can be transformed into cell linesusing expression vectors which may contain viral origins of replicationand/or endogenous expression elements and a selectable marker gene onthe same or on a separate vector. Following the introduction of thevector, cells may be allowed to grow for about 1 to 2 days in enrichedmedia before being switched to selective media. The purpose of theselectable marker is to confer resistance to a selective agent, and itspresence allows growth and recovery of cells which successfully expressthe introduced sequences. Resistant clones of stably transformed cellsmay be propagated using tissue culture techniques appropriate to thecell type.

[0097] Any number of selection systems may be used to recovertransformed cell lines. These include, but are not limited to, theherpes simplex virus thymidine kinase and adeninephosphoribosyltransferase genes, for use in tk⁻ or apr⁻ cells,respectively. (See, e.g., Wigler, M. et al. (1977) Cell 11:223-232; andLowy, I. et al. (1980) Cell 22:817-823.) Also, antimetabolite,antibiotic, or herbicide resistance can be used as the basis forselection. For example, dhfr confers resistance to methotrexate; neoconfers resistance to the aminoglycosides neomycin and G-418; and als orpat confer resistance to chlorsulfuron and phosphinotricinacetyltransferase, respectively. (See, e.g., Wigler, M. et al. (1980)Proc. Natl. Acad. Sci. 77:3567-3570; Colbere-Garapin, F. et al (1981) J.Mol. Biol. 150:1-14; and Murry, supra.) Additional selectable genes havebeen described, e.g., trpB and hisd, which alter cellular requirementsfor metabolites. (See, e.g., Hartman, S. C. and R. C. Mulligan (1988)Proc. Natl. Acad. Sci. 85:8047-8051.) Visible markers, e.g.,anthocyanins, green fluorescent proteins (GFP) (Clontech, Palo Alto,Calif.), β-glucuronidase and its substrate β-D-glucuronoside, orluciferase and its substrate luciferin may be used. These markers can beused not only to identify transformants, but also to quantify the amountof transient or stable protein expression attributable to a specificvector system. (See, e.g., Rhodes, C. A. et al. (1995) Methods Mol.Biol. 55:121-131.)

[0098] Although the presence/absence of marker gene expression suggeststhat the gene of interest is also present, the presence and expressionof the gene may need to be confirmed. For example, if the sequenceencoding DRPCS is inserted within a marker gene sequence, transformedcells containing sequences encoding DRPCS can be identified by theabsence of marker gene function. Alternatively, a marker gene can beplaced in tandem with a sequence encoding DRPCS under the control of asingle promoter. Expression of the marker gene in response to inductionor selection usually indicates expression of the tandem gene as well.

[0099] In general, host cells that contain the nucleic acid sequenceencoding DRPCS and that express DRPCS may be identified by a variety ofprocedures known to those of skill in the art. These procedures include,but are not limited to, DNA-DNA or DNA-RNA hybridizations, PCRamplification, and protein bioassay or immunoassay techniques whichinclude membrane, solution, or chip based technologies for the detectionand/or quantification of nucleic acid or protein sequences.

[0100] Immunological methods for detecting and measuring the expressionof DRPCS using either specific polyclonal or monoclonal antibodies areknown in the art. Examples of such techniques include enzyme-linkedimmunosorbent assays (ELISAs), radioimmunoassays (RIAs), andfluorescence activated cell sorting (FACS). A two-site, monoclonal-basedimmunoassay utilizing monoclonal antibodies reactive to twonon-interfering epitopes on DRPCS is preferred, but a competitivebinding assay may be employed. These and other assays are well known inthe art. (See, e.g., Hampton, R. et al. (1990) Serological Methods, aLaboratory Manual, APS Press, St Paul, Minn., Section IV; Coligan, J. E.et al. (1997 and periodic supplements) Current Protocols in Immunology,Greene Pub. Associates and Wiley-Interscience, New York, N.Y.; andMaddox, D. E. et al. (1983) J. Exp. Med. 158:1211-1216).

[0101] A wide variety of labels and conjugation techniques are known bythose skilled in the art and may be used in various nucleic acid andamino acid assays. Means for producing labeled hybridization or PCRprobes for detecting sequences related to polynucleotides encoding DRPCSinclude oligolabeling, nick translation, end-labeling, or PCRamplification using a labeled nucleotide. Alternatively, the sequencesencoding DRPCS, or any fragments thereof, may be cloned into a vectorfor the production of an mRNA probe. Such vectors are known in the art,are commercially available, and may be used to synthesize RNA probes invitro by addition of an appropriate RNA polymerase such as T7, T3, orSP6 and labeled nucleotides. These procedures may be conducted using avariety of commercially available kits, such as those provided byPharmacia & Upjohn (Kalamazoo, Mich.), Promega (Madison, Wis.), and U.S.Biochemical Corp. (Cleveland, Ohio). Suitable reporter molecules orlabels which may be used for ease of detection include radionuclides,enzymes, fluorescent, chemiluminescent, or chromogenic agents, as wellas substrates, cofactors, inhibitors, magnetic particles, and the like.

[0102] Host cells transformed with nucleotide sequences encoding DRPCSmay be cultured under conditions suitable for the expression andrecovery of the protein from cell culture. The protein produced by atransformed cell may be secreted or retained intracellularly dependingon the sequence and/or the vector used. As will be understood by thoseof skill in the art, expression vectors containing polynucleotides whichencode DRPCS may be designed to contain signal sequences which directsecretion of DRPCS through a prokaryotic or eukaryotic cell membrane.

[0103] In addition, a host cell strain may be chosen for its ability tomodulate expression of the inserted sequences or to process theexpressed protein in the desired fashion. Such modifications of thepolypeptide include, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation, and acylation.Post-translational processing which cleaves a “prepro” form of theprotein may also be used to specify protein targeting, folding, and/oractivity. Different host cells which have specific cellular machineryand characteristic mechanisms for post-translational activities (e.g.,CHO, HeLa, MDCK, HEK293, and W138), are available from the American TypeCulture Collection (ATCC, Manassas, Va.) and may be chosen to ensure thecorrect modification and processing of the foreign protein.

[0104] In another embodiment of the invention, natural, modified, orrecombinant nucleic acid sequences encoding DRPCS may be ligated to aheterologous sequence resulting in translation of a fusion protein inany of the aforementioned host systems. For example, a chimeric DRPCSprotein containing a heterologous moiety that can be recognized by acommercially available antibody may facilitate the screening of peptidelibraries for inhibitors of DRPCS activity. Heterologous protein andpeptide moieties may also facilitate purification of fusion proteinsusing commercially available affinity matrices. Such moieties include,but are not limited to, glutathione S-transferase (GST), maltose bindingprotein (MBP), thioredoxin (Trx), calmodulin binding peptide (CBP),6-His, FLAG, c-myc, and hemagglutinin (HA). GST, MBP, Trx, CBP, and6-His enable purification of their cognate fusion proteins onimmobilized glutathione, maltose, phenylarsine oxide, calmodulin, andmetal-chelate resins, respectively. FLAG, c-myc, and hemagglutinin (HA)enable immunoaffinity purification of fusion proteins using commerciallyavailable monoclonal and polyclonal antibodies that specificallyrecognize these epitope tags. A fusion protein may also be engineered tocontain a proteolytic cleavage site located between the DRPCS encodingsequence and the heterologous protein sequence, so that DRPCS may becleaved away from the heterologous moiety following purification.Methods for fusion protein expression and purification are discussed inAusubel, F. M. et al. (1995 and periodic supplements) Current Protocolsin Molecular Biology, John Wiley & Sons, New York, N.Y., ch 10. Avariety of commercially available kits may also be used to facilitateexpression and purification of fusion proteins.

[0105] In a further embodiment of the invention, synthesis ofradiolabeled DRPCS may be achieved in vitro using the TNT rabbitreticulocyte lysate or wheat germ extract systems (Promega, Madison,Wis.). These systems couple transcription and translation ofprotein-coding sequences operably associated with the T7, T3, or SP6promoters. Translation takes place in the presence of a radiolabeledamino acid precursor, preferably ³⁵S-methionine.

[0106] Fragments of DRPCS may be produced not only by recombinantproduction, but also by direct peptide synthesis using solid-phasetechniques. (See, e.g., Creighton, supra pp. 55-60.) Protein synthesismay be performed by manual techniques or by automation. Automatedsynthesis may be achieved, for example, using a 431A peptide synthesizer(Applied Biosystems, Foster City Calif.). Various fragments of DRPCS maybe synthesized separately and then combined to produce the full lengthmolecule.

THERAPEUTICS

[0107] Chemical and structural similarity exists among DRPCS, the humancardiac delayed rectifier potassium channel protein (hDRPC) (GI 452494)and the rat delayed rectifier potassium channel (rDRPC) (GI 203977). Inaddition, DRPCS is expressed in cancerous tissues and in cardiovasculartissues. Therefore, DRPCS appears to play a role in cancer,cardiovascular disorders, and neuronal disorders.

[0108] Therefore, in one embodiment, an antagonist of DRPCS may beadministered to a subject to treat or prevent a cancer. Such a cancermay include, but is not limited to, adenocarcinoma, leukemia, lymphoma,melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular, cancersof the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix,gall bladder, ganglia, gastrointestinal tract, heart, kidney, liver,lung, muscle, ovary, pancreas, parathyroid, penis, prostate, salivaryglands, skin, spleen, testis, thymus, thyroid, and uterus. In oneaspect, an antibody which specifically binds DRPCS may be used directlyas an antagonist or indirectly as a targeting or delivery mechanism forbringing a pharmaceutical agent to cells or tissue which express DRPCS.

[0109] In an additional embodiment, a vector expressing the complementof the polynucleotide encoding DRPCS may be administered to a subject totreat or prevent a cancer including, but not limited to, those describedabove.

[0110] In a further embodiment, DRPCS or a fragment or derivativethereof may be administered to a subject to treat or prevent acardiovascular disorder. Such disorders can include, but are not limitedto, arteriosclerosis including atherosclerosis and nonatheromatusarteriosclerosis, hypertension, stroke, coronary artery disease,ischemia, myocardial infarction, angina pectoris, cardiac arrhythmias,sinoatrial node blocks, atrioventricular node blocks, chronichemodynamic overload, aneurysm, Jervell and Lange-Nielsen syndrome, andlong QT syndrome.

[0111] In another embodiment, a vector capable of expressing DRPCS or afragment or derivative thereof may be administered to a subject to treator prevent a cardiovascular disorder including, but not limited to,those described above.

[0112] In a further embodiment, a pharmaceutical composition comprisinga substantially purified DRPCS in conjunction with a suitablepharmaceutical carrier may be administered to a subject to treat orprevent a cardiovascular disorder including, but not limited to, thoseprovided above.

[0113] In still another embodiment, an agonist which modulates theactivity of DRPCS may be administered to a subject to treat or prevent acardiovascular disorder including, but not limited to, those listedabove.

[0114] In yet another embodiment, an antagonist of DRPCS may beadministered to a subject to treat or prevent a neuronal disorder. Sucha disorder may include, but is not limited to, akathesia, Alzheimer'sdisease, amnesia, amyotrophic lateral sclerosis, bipolar disorder,catatonia, cerebral neoplasms, dementia, depression, diabeticneuropathy, Down's syndrome, tardive dyskinesia, dystonias, epilepsy,Huntington's disease, peripheral neuropathy, multiple sclerosis,neurofibromatosis, Parkinson's disease, paranoid psychoses, postherpeticneuralgia, schizophrenia, and Tourette's disorder. In one aspect, anantibody which specifically binds DRPCS may be used directly as anantagonist or indirectly as a targeting or delivery mechanism forbringing a pharmaceutical agent to cells or tissue which express DRPCS.

[0115] In an additional embodiment, a vector expressing the complementof the polynucleotide encoding DRPCS may be administered to a subject totreat or prevent a neuronal disorder including, but not limited to,those described above.

[0116] In other embodiments, any of the proteins, antagonists,antibodies, agonists, complementary sequences, or vectors of theinvention may be administered in combination with other appropriatetherapeutic agents. Selection of the appropriate agents for use incombination therapy may be made by one of ordinary skill in the art,according to conventional pharmaceutical principles. The combination oftherapeutic agents may act synergistically to effect the treatment orprevention of the various disorders described above. Using thisapproach, one may be able to achieve therapeutic efficacy with lowerdosages of each agent, thus reducing the potential for adverse sideeffects.

[0117] An antagonist of DRPCS may be produced using methods which aregenerally known in the art. In particular, purified DRPCS may be used toproduce antibodies or to screen libraries of pharmaceutical agents toidentify those which specifically bind DRPCS. Antibodies to DRPCS mayalso be generated using methods that are well known in the art. Suchantibodies may include, but are not limited to, polyclonal, monoclonal,chimeric, and single chain antibodies, Fab fragments, and fragmentsproduced by a Fab expression library. Neutralizing antibodies (i.e.,those which inhibit dimer formation) are especially preferred fortherapeutic use.

[0118] For the production of antibodies, various hosts including goats,rabbits, rats, mice, humans, and others may be immunized by injectionwith DRPCS or with any fragment or oligopeptide thereof which hasimmunogenic properties. Depending on the host species, various adjuvantsmay be used to increase immunological response. Such adjuvants include,but are not limited to, Freund's, mineral gels such as aluminumhydroxide, and surface active substances such as lysolecithin, pluronicpolyols, polyanions, peptides, oil emulsions, KLH, and dinitrophenol.Among adjuvants used in humans, BCG (bacilli Calmette-Guerin) andCorynebacterium parvum are especially preferable.

[0119] It is preferred that the oligopeptides, peptides, or fragmentsused to induce antibodies to DRPCS have an amino acid sequenceconsisting of at least about 5 amino acids, and, more preferably, of atleast about 10 amino acids. It is also preferable that theseoligopeptides, peptides, or fragments are identical to a portion of theamino acid sequence of the natural protein and contain the entire aminoacid sequence of a small, naturally occurring molecule. Short stretchesof DRPCS amino acids may be fused with those of another protein, such asKLH, and antibodies to the chimeric molecule may be produced.

[0120] Monoclonal antibodies to DRPCS may be prepared using anytechnique which provides for the production of antibody molecules bycontinuous cell lines in culture. These include, but are not limited to,the hybridoma technique, the human B-cell hybridoma technique, and theEBV-hybridoma technique. (See, e.g., Kohler, G. et al. (1975) Nature256:495-497; Kozbor, D. et al. (1985) J. Immunol. Methods 81:31-42;Cote, R. J. et al. (1983) Proc. Natl. Acad. Sci. 80:2026-2030; and Cole,S. P. et al. (1984) Mol. Cell Biol. 62:109-120.)

[0121] In addition, techniques developed for the production of “chimericantibodies,” such as the splicing of mouse antibody genes to humanantibody genes to obtain a molecule with appropriate antigen specificityand biological activity, can be used. (See, e.g., Morrison, S. L. et al.(1984) Proc. Natl. Acad. Sci. 81:6851-6855; Neuberger, M. S. et al.(1984) Nature 312:604-608; and Takeda, S. et al. (1985) Nature314:452-454.) Alternatively, techniques described for the production ofsingle chain antibodies may be adapted, using methods known in the art,to produce DRPCS-specific single chain antibodies. Antibodies withrelated specificity, but of distinct idiotypic composition, may begenerated by chain shuffling from random combinatorial immunoglobulinlibraries. (See, e.g., Burton D. R. (1991) Proc. Natl. Acad. Sci.88:10134-10137.)

[0122] Antibodies may also be produced by inducing in vivo production inthe lymphocyte population or by screening immunoglobulin libraries orpanels of highly specific binding reagents as disclosed in theliterature. (See, e.g., Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci.86: 3833-3837; and Winter, G. et al. (1991) Nature 349:293-299.)

[0123] Antibody fragments which contain specific binding sites for DRPCSmay also be generated. For example, such fragments include, but are notlimited to, F(ab′)2 fragments produced by pepsin digestion of theantibody molecule and Fab fragments generated by reducing the disulfidebridges of the F(ab′)2 fragments. Alternatively, Fab expressionlibraries may be constructed to allow rapid and easy identification ofmonoclonal Fab fragments with the desired specificity. (See, e.g., Huse,W. D. et al: (1989) Science 246:1275-1281.)

[0124] Various immunoassays may be used for screening to identifyantibodies having the desired specificity. Numerous protocols forcompetitive binding or immunoradiometric assays using either polyclonalor monoclonal antibodies with established specificities are well knownin the art. Such immunoassays typically involve the measurement ofcomplex formation between DRPCS and its specific antibody. A two-site,monoclonal-based immunoassay utilizing monoclonal antibodies reactive totwo non-interfering DRPCS epitopes is preferred, but a competitivebinding assay may also be employed. (Maddox, supra.)

[0125] In another embodiment of the invention, the polynucleotidesencoding DRPCS, or any fragment or complement thereof, may be used fortherapeutic purposes. In one aspect, the complement of thepolynucleotide encoding DRPCS may be used in situations in which itwould be desirable to block the transcription of the mRNA. Inparticular, cells may be transformed with sequences complementary topolynucleotides encoding DRPCS. Thus, complementary molecules orfragments may be used to modulate DRPCS activity, or to achieveregulation of gene function. Such technology is now well known in theart, and sense or antisense oligonucleotides or larger fragments can bedesigned from various locations along the coding or control regions ofsequences encoding DRPCS.

[0126] Expression vectors derived from retroviruses, adenoviruses, orherpes or vaccinia viruses, or from various bacterial plasmids, may beused for delivery of nucleotide sequences to the targeted organ, tissue,or cell population. Methods which are well known to those skilled in theart can be used to construct vectors to express nucleic acid sequencescomplementary to the polynucleotides encoding DRPCS. (See, e.g.,Sambrook, supra; and Ausubel, supra.)

[0127] Genes encoding DRPCS can be turned off by transforming a cell ortissue with expression vectors which express high levels of apolynucleotide, or fragment thereof, encoding DRPCS. Such constructs maybe used to introduce untranslatable sense or antisense sequences into acell. Even in the absence of integration into the DNA, such vectors maycontinue to transcribe RNA molecules until they are disabled byendogenous nucleases. Transient expression may last for a month or morewith a non-replicating vector, and may last even longer if appropriatereplication elements are part of the vector system.

[0128] As mentioned above, modifications of gene expression can beobtained by designing complementary sequences or antisense molecules(DNA, RNA, or PNA) to the control, 5′, or regulatory regions of the geneencoding DRPCS. Oligonucleotides derived from the transcriptioninitiation site, e.g., between about positions −10 and +10 from thestart site, are preferred. Similarly, inhibition can be achieved usingtriple helix base-pairing methodology. Triple helix pairing is usefulbecause it causes inhibition of the ability of the double helix to opensufficiently for the binding of polymerases, transcription factors, orregulatory molecules. Recent therapeutic advances using triplex DNA havebeen described in the literature. (See, e.g., Gee, J. E. et al. (1994)in Huber, B. E. and B. I. Carr, Molecular and Immunologic Approaches,Futura Publishing Co., Mt. Kisco, N.Y., pp. 163-177.) A complementarysequence or antisense molecule may also be designed to block translationof mRNA by preventing the transcript from binding to ribosomes.

[0129] Ribozymes, enzymatic RNA molecules, may also be used to catalyzethe specific cleavage of RNA. The mechanism of ribozyme action involvessequence-specific hybridization of the ribozyme molecule tocomplementary target RNA, followed by endonucleolytic cleavage. Forexample, engineered hammerhead motif ribozyme molecules may specificallyand efficiently catalyze endonucleolytic cleavage of sequences encodingDRPCS.

[0130] Specific ribozyme cleavage sites within any potential RNA targetare initially identified by scanning the target molecule for ribozymecleavage sites, including the following sequences: GUA, GUU, and GUC.Once identified, short RNA sequences of between 15 and 20ribonucleotides, corresponding to the region of the target genecontaining the cleavage site, may be evaluated for secondary structuralfeatures which may render the oligonucleotide inoperable. Thesuitability of candidate targets may also be evaluated by testingaccessibility to hybridization with complementary oligonucleotides usingribonuclease protection assays.

[0131] Complementary ribonucleic acid molecules and ribozymes of theinvention may be prepared by any method known in the art for thesynthesis of nucleic acid molecules. These include techniques forchemically synthesizing oligonucleotides such as solid phasephosphoramidite chemical synthesis. Alternatively, RNA molecules may begenerated by in vitro and in vivo transcription of DNA sequencesencoding DRPCS. Such DNA sequences may be incorporated into a widevariety of vectors with suitable RNA polymerase promoters such as T7 orSP6. Alternatively, these cDNA constructs that synthesize complementaryRNA, constitutively or inducibly, can be introduced into cell lines,cells, or tissues.

[0132] RNA molecules may be modified to increase intracellular stabilityand half-life. Possible modifications include, but are not limited to,the addition of flanking sequences at the 5′ and/or 3′ ends of themolecule, or the use of phosphorothioate or 2′ O-methyl rather thanphosphodiesterase linkages within the backbone of the molecule. Thisconcept is inherent in the production of PNAs and can be extended in allof these molecules by the inclusion of nontraditional bases such asinosine, queosine, and wybutosine, as well as acetyl-, methyl-, thio-,and similarly modified forms of adenine, cytidine, guanine, thymine, anduridine which are not as easily recognized by endogenous endonucleases.

[0133] Many methods for introducing vectors into cells or tissues areavailable and equally suitable for use in vivo, in vitro, and ex vivo.For ex vivo therapy, vectors may be introduced into stem cells takenfrom the patient and clonally propagated for autologous transplant backinto that same patient. Delivery by transfection, by liposomeinjections, or by polycationic amino polymers may be achieved usingmethods which are well known in the art. (See, e.g., Goldman, C. K. etal. (1997) Nature Biotechnology 15:462-466.)

[0134] Any of the therapeutic methods described above may be applied toany subject in need of such therapy, including, for example, mammalssuch as dogs, cats, cows, horses, rabbits, monkeys, and most preferably,humans.

[0135] An additional embodiment of the invention relates to theadministration of a pharmaceutical or sterile composition, inconjunction with a pharmaceutically acceptable carrier, for any of thetherapeutic effects discussed above. Such pharmaceutical compositionsmay consist of DRPCS, antibodies to DRPCS, and mimetics, agonists,antagonists, or inhibitors of DRPCS. The compositions may beadministered alone or in combination with at least one other agent, suchas a stabilizing compound, which may be administered in any sterile,biocompatible pharmaceutical carrier including, but not limited to,saline, buffered saline, dextrose, and water. The compositions may beadministered to a patient alone, or in combination with other agents,drugs, or hormones.

[0136] The pharmaceutical compositions utilized in this invention may beadministered by any number of routes including, but not limited to,oral, intravenous, intramuscular, intra-arterial, intramedullary,intrathecal, intraventricular, transdermal, subcutaneous,intraperitoneal, intranasal, enteral, topical, sublingual, or rectalmeans.

[0137] In addition to the active ingredients, these pharmaceuticalcompositions may contain suitable pharmaceutically-acceptable carrierscomprising excipients and auxiliaries which facilitate processing of theactive compounds into preparations which can be used pharmaceutically.Further details on techniques for formulation and administration may befound in the latest edition of Remington's Pharmaceutical Sciences(Maack Publishing Co., Easton, Pa.).

[0138] Pharmaceutical compositions for oral administration can beformulated using pharmaceutically acceptable carriers well known in theart in dosages suitable for oral administration. Such carriers enablethe pharmaceutical compositions to be formulated as tablets, pills,dragees, capsules, liquids, gels, syrups, slurries, suspensions, and thelike, for ingestion by the patient.

[0139] Pharmaceutical preparations for oral use can be obtained throughcombining active compounds with solid excipient and processing theresultant mixture of granules (optionally, after grinding) to obtaintablets or dragee cores. Suitable auxiliaries can be added, if desired.Suitable excipients include carbohydrate or protein fillers, such assugars, including lactose, sucrose, mannitol, and sorbitol; starch fromcorn, wheat, rice, potato, or other plants; cellulose, such as methylcellulose, hydroxypropylmethyl-cellulose, or sodiumcarboxymethylcellulose; gums, including arabic and tragacanth; andproteins, such as gelatin and collagen. If desired, disintegrating orsolubilizing agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, and alginic acid or a salt thereof, such as sodiumalginate.

[0140] Dragee cores may be used in conjunction with suitable coatings,such as concentrated sugar solutions, which may also contain gum arabic,talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/ortitanium dioxide, lacquer solutions, and suitable organic solvents orsolvent mixtures. Dyestuffs or pigments may be added to the tablets ordragee coatings for product identification or to characterize thequantity of active compound, i.e., dosage.

[0141] Pharmaceutical preparations which can be used orally includepush-fit capsules made of gelatin, as well as soft, sealed capsules madeof gelatin and a coating, such as glycerol or sorbitol. Push-fitcapsules can contain active ingredients mixed with fillers or binders,such as lactose or starches, lubricants, such as talc or magnesiumstearate, and, optionally, stabilizers. In soft capsules, the activecompounds may be dissolved or suspended in suitable liquids, such asfatty oils, liquid, or liquid polyethylene glycol with or withoutstabilizers.

[0142] Pharmaceutical formulations suitable for parenteraladministration may be formulated in aqueous solutions, preferably inphysiologically compatible buffers such as Hanks' solution, Ringer'ssolution, or physiologically buffered saline. Aqueous injectionsuspensions may contain substances which increase the viscosity of thesuspension, such as sodium carboxymethyl cellulose, sorbitol, ordextran. Additionally, suspensions of the active compounds may beprepared as appropriate oily injection suspensions. Suitable lipophilicsolvents or vehicles include fatty oils, such as sesame oil, orsynthetic fatty acid esters, such as ethyl oleate, triglycerides, orliposomes. Non-lipid polycationic amino polymers may also be used fordelivery. Optionally, the suspension may also contain suitablestabilizers or agents to increase the solubility of the compounds andallow for the preparation of highly concentrated solutions.

[0143] For topical or nasal administration, penetrants appropriate tothe particular barrier to be permeated are used in the formulation. Suchpenetrants are generally known in the art.

[0144] The pharmaceutical compositions of the present invention may bemanufactured in a manner that is known in the art, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping, or lyophilizing processes.

[0145] The pharmaceutical composition may be provided as a salt and canbe formed with many acids, including but not limited to, hydrochloric,sulfuric, acetic, lactic, tartaric, malic, and succinic acid. Salts tendto be more soluble in aqueous or other protonic solvents than are thecorresponding free base forms. In other cases, the preferred preparationmay be a lyophilized powder which may contain any or all of thefollowing: 1 mM to 50 mM histidine, 0.1% to 2% sucrose, and 2% to 7%mannitol, at a pH range of 4.5 to 5.5, that is combined with bufferprior to use.

[0146] After pharmaceutical compositions have been prepared, they can beplaced in an appropriate container and labeled for treatment of anindicated condition. For administration of DRPCS, such labeling wouldinclude amount, frequency, and method of administration.

[0147] Pharmaceutical compositions suitable for use in the inventioninclude compositions wherein the active ingredients are contained in aneffective amount to achieve the intended purpose. The determination ofan effective dose is well within the capability of those skilled in theart.

[0148] For any compound, the therapeutically effective dose can beestimated initially either in cell culture assays, e.g., of neoplasticcells or in animal models such as mice, rats, rabbits, dogs, or pigs. Ananimal model may also be used to determine the appropriate concentrationrange and route of administration. Such information can then be used todetermine useful doses and routes for administration in humans.

[0149] A therapeutically effective dose refers to that amount of activeingredient, for example DRPCS or fragments thereof, antibodies of DRPCS,and agonists, antagonists or inhibitors of DRPCS, which ameliorates thesymptoms or condition. Therapeutic efficacy and toxicity may bedetermined by standard pharmaceutical procedures in cell cultures orwith experimental animals, such as by calculating the ED₅₀ (the dosetherapeutically effective in 50% of the population) or LD₅₀ (the doselethal to 50% of the population) statistics. The dose ratio of toxic totherapeutic effects is the therapeutic index, and it can be expressed asthe LD₅₀/ED₅₀ ratio. Pharmaceutical compositions which exhibit largetherapeutic indices are preferred. The data obtained from cell cultureassays and animal studies are used to formulate a range of dosage forhuman use. The dosage contained in such compositions is preferablywithin a range of circulating concentrations that includes the ED₅₀ withlittle or no toxicity. The dosage varies within this range dependingupon the dosage form employed, the sensitivity of the patient, and theroute of administration.

[0150] The exact dosage will be determined by the practitioner, in lightof factors related to the subject requiring treatment. Dosage andadministration are adjusted to provide sufficient levels of the activemoiety or to maintain the desired effect. Factors which may be takeninto account include the severity of the disease state, the generalhealth of the subject, the age, weight, and gender of the subject, timeand frequency of administration, drug combination(s), reactionsensitivities, and response to therapy. Long-acting pharmaceuticalcompositions may be administered every 3 to 4 days, every week, orbiweekly depending on the half-life and clearance rate of the particularformulation.

[0151] Normal dosage amounts may vary from about 0.1 μg to 100,000 μg,up to a total dose of about 1 gram, depending upon the route ofadministration. Guidance as to particular dosages and methods ofdelivery is provided in the literature and generally available topractitioners in the art. Those skilled in the art will employ differentformulations for nucleotides than for proteins or their inhibitors.Similarly, delivery of polynucleotides or polypeptides will be specificto particular cells, conditions, locations, etc.

DIAGNOSTICS

[0152] In another embodiment, antibodies which specifically bind DRPCSmay be used for the diagnosis of disorders characterized by expressionof DRPCS, or in assays to monitor patients being treated with DRPCS oragonists, antagonists, or inhibitors of DRPCS. Antibodies useful fordiagnostic purposes may be prepared in the same manner as describedabove for therapeutics. Diagnostic assays for DRPCS include methodswhich utilize the antibody and a label to detect DRPCS in human bodyfluids or in extracts of cells or tissues. The antibodies may be usedwith or without modification, and may be labeled by covalent ornon-covalent attachment of a reporter molecule. A wide variety ofreporter molecules, several of which are described above, are known inthe art and may be used.

[0153] A variety of protocols for measuring DRPCS, including ELISAs,RIAs, and FACS, are known in the art and provide a basis for diagnosingaltered or abnormal levels of DRPCS expression. Normal or standardvalues for DRPCS expression are established by combining body fluids orcell extracts taken from normal mammalian subjects, preferably human,with antibody to DRPCS under conditions suitable for complex formationThe amount of standard complex formation may be quantitated by variousmethods, preferably by photometric means. Quantities of DRPCS expressedin subject, control, and disease samples from biopsied tissues arecompared with the standard values. Deviation between standard andsubject values establishes the parameters for diagnosing disease.

[0154] In another embodiment of the invention, the polynucleotidesencoding DRPCS may be used for diagnostic purposes. The polynucleotideswhich may be used include oligonucleotide sequences, complementary RNAand DNA molecules, and PNAs. The polynucleotides may be used to detectand quantitate gene expression in biopsied tissues in which expressionof DRPCS may be correlated with disease. The diagnostic assay may beused to determine absence, presence, and excess expression of DRPCS, andto monitor regulation of DRPCS levels during therapeutic intervention.

[0155] In one aspect, hybridization with PCR probes which are capable ofdetecting polynucleotide sequences, including genomic sequences,encoding DRPCS or closely related molecules may be used to identifynucleic acid sequences which encode DRPCS. The specificity of the probe,whether it is made from a highly specific region, e.g., the 5′regulatory region, or from a less specific region, e.g., a conservedmotif, and the stringency of the hybridization or amplification(maximal, high, intermediate, or low), will determine whether the probeidentifies only naturally occurring sequences encoding DRPCS, allelicvariants, or related sequences.

[0156] Probes may also be used for the detection of related sequences,and should preferably have at least 50% sequence identity to any of theDRPCS encoding sequences. The hybridization probes of the subjectinvention may be DNA or RNA and may be derived from the sequence of SEQID NO:2, or from genomic sequences including promoters, enhancers, andintrons of the DRPCS gene.

[0157] Means for producing specific hybridization probes for DNAsencoding DRPCS include the cloning of polynucleotide sequences encodingDRPCS or DRPCS derivatives into vectors for the production of mRNAprobes. Such vectors are known in the art, are commercially available,and may be used to synthesize RNA probes in vitro by means of theaddition of the appropriate RNA polymerases and the appropriate labelednucleotides. Hybridization probes may be labeled by a variety ofreporter groups, for example, by radionuclides such as ³²P or ³⁵S, or byenzymatic labels, such as alkaline phosphatase coupled to the probe viaavidin/biotin coupling systems, and the like.

[0158] Polynucleotide sequences encoding DRPCS may be used for thediagnosis of a disorder associated with expression of DRPCS. Examples ofsuch a disorder include, but are not limited to, cancers such asadenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma,teratocarcinoma, and, in particular, cancers of the adrenal gland,bladder, bone, bone marrow, brain, breast, cervix, gall bladder,ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle,ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin,spleen, testis, thymus, thyroid, and uterus, cardiovascular diseasessuch as arteriosclerosis including atherosclerosis and nonatheromatusarteriosclerosis, hypertension, stroke, coronary artery disease,ischemia, myocardial infarction, angina pectoris, cardiac arrhythmias,sinoatrial node blocks, atrioventricular node blocks, chronichemodynamic overload, aneurysm, Jervell and Lange-Nielsen syndrome, andlong QT syndrome, and neuronal disorders such as akathesia, Alzheimer'sdisease, amnesia, amyotrophic lateral sclerosis, bipolar disorder,catatonia, cerebral neoplasms, dementia, depression, diabeticneuropathy, Down's syndrome, tardive dyskinesia, dystonias, epilepsy,Huntington's disease, peripheral neuropathy, multiple sclerosis,neurofibromatosis, Parkinson's disease, paranoid psychoses, postherpeticneuralgia, schizophrenia, and Tourette's disorder. The polynucleotidesequences encoding DRPCS may be used in Southern or Northern analysis,dot blot, or other membrane-based technologies; in PCR technologies; indipstick, pin, and ELISA assays; and in microarrays utilizing fluids ortissues from patients to detect altered DRPCS expression. Suchqualitative or quantitative methods are well known in the art.

[0159] In a particular aspect, the nucleotide sequences encoding DRPCSmay be useful in assays that detect the presence of associateddisorders, particularly those mentioned above. The nucleotide sequencesencoding DRPCS may be labeled by standard methods and added to a fluidor tissue sample from a patient under conditions suitable for theformation of hybridization complexes. After a suitable incubationperiod, the sample is washed and the signal is quantitated and comparedwith a standard value. If the amount of signal in the patient sample issignificantly altered in comparison to a control sample then thepresence of altered levels of nucleotide sequences encoding DRPCS in thesample indicates the presence of the associated disorder. Such assaysmay also be used to evaluate the efficacy of a particular therapeutictreatment regimen in animal studies, in clinical trials, or to monitorthe treatment of an individual patient.

[0160] In order to provide a basis for the diagnosis of a disorderassociated with expression of DRPCS, a normal or standard profile forexpression is established. This may be accomplished by combining bodyfluids or cell extracts taken from normal subjects, either animal orhuman, with a sequence, or a fragment thereof, encoding DRPCS, underconditions suitable for hybridization or amplification. Standardhybridization may be quantified by comparing the values obtained fromnormal subjects with values from an experiment in which a known amountof a substantially purified polynucleotide is used. Standard valuesobtained in this manner may be compared with values obtained fromsamples from patients who are symptomatic for a disorder. Deviation fromstandard values is used to establish the presence of a disorder.

[0161] Once the presence of a disorder is established and a treatmentprotocol is initiated, hybridization assays may be repeated on a regularbasis to determine if the level of expression in the patient begins toapproximate that which is observed in the normal subject. The resultsobtained from successive assays may be used to show the efficacy oftreatment over a period ranging from several days to months.

[0162] With respect to cancer, the presence of a relatively high amountof transcript in biopsied tissue from an individual may indicate apredisposition for the development of the disease, or may provide ameans for detecting the disease prior to the appearance of actualclinical symptoms. A more definitive diagnosis of this type may allowhealth professionals to employ preventative measures or aggressivetreatment earlier thereby preventing the development or furtherprogression of the cancer.

[0163] Additional diagnostic uses for oligonucleotides designed from thesequences encoding DRPCS may involve the use of PCR. These oligomers maybe chemically synthesized, generated enzymatically, or produced invitro. Oligomers will preferably contain a fragment of a polynucleotideencoding DRPCS, or a fragment of a polynucleotide complementary to thepolynucleotide encoding DRPCS, and will be employed under optimizedconditions for identification of a specific gene or condition. Oligomersmay also be employed under less stringent conditions for detection orquantitation of closely related DNA or RNA sequences.

[0164] Methods which may also be used to quantitate the expression ofDRPCS include radiolabeling or biotinylating nucleotides,coamplification of a control nucleic acid, and interpolating resultsfrom standard curves. (See, e.g., Melby, P. C. et al. (1993) J. Immunol.Methods 159:235-244; and Duplaa, C. et al. (1993) Anal. Biochem.229-236.) The speed of quantitation of multiple samples may beaccelerated by running the assay in an ELISA format where the oligomerof interest is presented in various dilutions and a spectrophotometricor calorimetric response gives rapid quantitation.

[0165] In further embodiments, oligonucleotides or longer fragmentsderived from any of the polynucleotide sequences described herein may beused as targets in a microarray. The microarray can be used to monitorthe expression level of large numbers of genes simultaneously and toidentify genetic variants, mutations, and polymorphisms. Thisinformation may be used to determine gene function, to understand thegenetic basis of a disorder, to diagnose a disorder, and to develop andmonitor the activities of therapeutic agents.

[0166] Microarrays may be prepared, used, and analyzed using methodsknown in the art. (See, e.g., Brennan, T. M. et al. (1995) U.S. Pat. No.5,474,796; Schena, M. et al. (1996) Proc. Natl. Acad. Sci.93:10614-10619; Baldeschweiler et al. (1995) PCT applicationWO95/251116; Shalon, D. et al. (1995) PCT application WO95/35505;Heller, R. A. et al. (1997) Proc. Natl. Acad. Sci. 94:2150-2155; andHeller, M. J. et al. (1997) U.S. Pat. No. 5,605,662.)

[0167] In another embodiment of the invention, nucleic acid sequencesencoding DRPCS may be used to generate hybridization probes useful inmapping the naturally occurring genomic sequence. The sequences may bemapped to a particular chromosome, to a specific region of a chromosome,or to artificial chromosome constructions, e.g., human artificialchromosomes (HACs), yeast artificial chromosomes (YACs), bacterialartificial chromosomes (BACs), bacterial P1 constructions, or singlechromosome cDNA libraries. (See, e.g., Price, C. M. (1993) Blood Rev.7:127-134; and Trask, B. J. (1991) Trends Genet. 7:149-154.)

[0168] Fluorescent in situ hybridization (FISH) may be correlated withother physical chromosome mapping techniques and genetic map data. (See,e.g., Heinz-Ulrich, et al. (1995) in Meyers, R. A. (ed.) MolecularBiology and Biotechnology, VCH Publishers New York, N.Y., pp. 965-968.)Examples of genetic map data can be found in various scientific journalsor at the Online Mendelian Inheritance in Man (OMIM) site. Correlationbetween the location of the gene encoding DRPCS on a physicalchromosomal map and a specific disorder, or a predisposition to aspecific disorder, may help define the region of DNA associated withthat disorder. The nucleotide sequences of the invention may be used todetect differences in gene sequences among normal, carrier, and affectedindividuals.

[0169] In situ hybridization of chromosomal preparations and physicalmapping techniques, such as linkage analysis using establishedchromosomal markers, may be used for extending genetic maps. Often theplacement of a gene on the chromosome of another mammalian species, suchas mouse, may reveal associated markers even if the number or arm of aparticular human chromosome is not known. New sequences can be assignedto chromosomal arms by physical mapping. This provides valuableinformation to investigators searching for disease genes usingpositional cloning or other gene discovery techniques. Once the diseaseor syndrome has been crudely localized by genetic linkage to aparticular genomic region, e.g., ataxia-telangiectasia to 11q22-23, anysequences mapping to that area may represent associated or regulatorygenes for further investigation. (See, e.g., Gatti, R. A. et al. (1988)Nature 336:577-580.) The nucleotide sequence of the subject inventionmay also be used to detect differences in the chromosomal location dueto translocation, inversion, etc., among normal, carrier, or affectedindividuals.

[0170] In another embodiment of the invention, DRPCS, its catalytic orimmunogenic fragments, or oligopeptides thereof can be used forscreening libraries of compounds in any of a variety of drug screeningtechniques. The fragment employed in such screening may be free insolution, affixed to a solid support, borne on a cell surface, orlocated intracellularly. The formation of binding complexes betweenDRPCS and the agent being tested may be measured.

[0171] Another technique for drug screening provides for high throughputscreening of compounds having suitable binding affinity to the proteinof interest. (See, e.g., Geysen, et al. (1984) PCT applicationWO84/03564.) In this method, large numbers of different small testcompounds are synthesized on a solid substrate, such as plastic pins orsome other surface. The test compounds are reacted with DRPCS, orfragments thereof, and washed. Bound DRPCS is then detected by methodswell known in the art. Purified DRPCS can also be coated directly ontoplates for use in the aforementioned drug screening techniques.Alternatively, non-neutralizing antibodies can be used to capture thepeptide and immobilize it on a solid support.

[0172] In another embodiment, one may use competitive drug screeningassays in which neutralizing antibodies capable of binding DRPCSspecifically compete with a test compound for binding DRPCS. In thismanner, antibodies can be used to detect the presence of any peptidewhich shares one or more antigenic determinants with DRPCS.

[0173] In additional embodiments, the nucleotide sequences which encodeDRPCS may be used in any molecular biology techniques that have yet tobe developed, provided the new techniques rely on properties ofnucleotide sequences that are currently known, including, but notlimited to, such properties as the triplet genetic code and specificbase pair interactions.

[0174] The examples below are provided to illustrate the subjectinvention and are not included for the purpose of limiting theinvention.

EXAMPLES

[0175] I. BRSTNOT03 cDNA Library Construction

[0176] The BRSTNOT03 cDNA library was constructed using polyA RNAisolated from non-tumorous breast tissue removed from a 54-year-oldCaucasian female during a bilateral radical mastectomy. Pathology forthe associated tumor tissue indicated residual invasive grade 3 mammaryductal adenocarcinoma. The remaining breast parenchyma exhibitedproliferative fibrocystic changes without atypia. Fibroadipose tissuefrom the right breast was negative for tumor. One of 10 axillary lymphnodes had a metastatic tumor as a microscopic intranodal focus. Familyhistory included a malignant neoplasm of the colon.

[0177] The frozen tissue was homogenized and lysed using a PT-3000homogenizer polytron (Brinkmann Instruments, Westbury, N.Y.) inguanidinium isothiocyanate solution. Lysates were then loaded on a 5.7 MCsCl cushion and ultracentrifuged in an SW28 swinging bucket rotor for18 hours at 25,000 rpm at ambient temperature. The RNA was extractedonce with acid phenol at pH 4.0 and once with phenol chloroform at pH8.0 and precipitated using 0.3 M sodium acetate and 2.5 volumes ofethanol, resuspended in DEPC-treated water and DNase treated for 25 minat 37° C. The reaction was stopped with an equal volume of acid phenol,and the RNA was isolated using the OLIGOTEX mRNA purification kit(QIAGEN, Chatsworth, Calif.) and used to construct the cDNA library.

[0178] Poly(A+) RNA was used for cDNA synthesis and construction of thecDNA library according to the recommended protocols in the SUPERSCRIPTplasmid system (GIBCO BRL). The cDNAs were fractionated on a SEPHAROSECL4B column (Pharmacia, Piscataway, N.J.), and those cDNAs exceeding 400bp were ligated into the PSPORT1 plasmid (GIBCO BRL). The recombinantplasmids were subsequently transformed into DH5a competent cells (GIBCOBRL).

[0179] II Isolation and Sequencing of cDNA Clones

[0180] Plasmid DNA was released from the cells and purified using theAGTC miniprep purification kit (Advanced Genetic TechnologiesCorporation, Gaithersburg, Md.). This kit consists of a 96 well blockwith reagents for 960 purifications. The recommended protocol wasemployed except for the following changes: 1) the 96 wells were eachfilled with only 1 ml of sterile Terrific Broth (GIBCO BRL) withcarbenicillin at 25 mg/L and glycerol at 0.4%; 2) the bacteria werecultured for 24 hours after the wells were inoculated and then lysedwith 60 μl of lysis buffer; 3) a centrifugation step employing theBeckman GS-6R at 2900 rpm for 5 min was performed before the contents ofthe block were added to the primary filter plate; and 4) the optionalstep of adding isopropanol to TRIS buffer was not routinely performed.After the last step in the protocol, samples were transferred to aBeckman 96-well block for storage.

[0181] The cDNAs were sequenced by the method of Sanger, F. and A. R.Coulson (1975; J. Mol. Biol. 94:441f), using a MICROLAB 2200 liquidtransfer system (Hamilton, Reno, Nev.) in combination with four PTC200thermal cyclers (MJ Research, Watertown, Mass.) and the 377 or 373 DNAsequencing systems (Applied Biosystems).

[0182] III. Similarity Searching of cDNA Clones and Their DeducedProteins

[0183] The nucleotide sequences and/or amino acid sequences of theSequence Listing were used to query sequences in the GenBank, SwissProt,BLOCKS, and Pima II databases. These databases, which contain previouslyidentified and annotated sequences, were searched for regions ofsimilarity using BLAST (Basic Local Alignment Search Tool). (See, e.g.,Altschul, S. F. (1993) J. Mol. Evol 36:290-300; and Altschul et al.(1990) J. Mol. Biol. 215:403-410.)

[0184] BLAST produced alignments of both nucleotide and amino acidsequences to determine sequence similarity. Because of the local natureof the alignments, BLAST was especially useful in determining exactmatches or in identifying homologs which may be of prokaryotic(bacterial) or eukaryotic (animal, fungal, or plant) origin. Otheralgorithms could have been used when dealing with primary sequencepatterns and secondary structure gap penalties. (See, e.g., Smith, T. etal. (1992) Protein Engineering 5:35-51.) The sequences disclosed in thisapplication have lengths of at least 49 nucleotides and have no morethan 12% uncalled bases (where N is recorded rather than A, C, G, or T).

[0185] The BLAST approach searched for matches between a query sequenceand a database sequence. BLAST evaluated the statistical significance ofany matches found, and reported only those matches that satisfy theuser-selected threshold of significance. In this application, thresholdwas set at 10⁻²⁵ for nucleotides and 10⁻⁸ for peptides.

[0186] Incyte nucleotide sequences were searched against the GenBankdatabases for primate (pri), rodent (rod), and other mammalian sequences(mam), and deduced amino acid sequences from the same clones were thensearched against GenBank functional protein databases, mammalian (mamp),vertebrate (vrtp), and eukaryote (eukp), for similarity.

[0187] Additionally, sequences identified from cDNA libraries may beanalyzed to identify those gene sequences encoding conserved proteinmotifs using an appropriate analysis program, e.g., BLOCKS. BLOCKS is aweighted matrix analysis algorithm based on short amino acid segments,or blocks, compiled from the PROSITE database. (Bairoch, A. et al.(1997) Nucleic Acids Res. 25:217-221.) The BLOCKS algorithm is usefulfor classifying genes with unknown functions. (Henikoff, S. and G. J.Henikoff (1991) Nucleic Acids Research 19:6565-6572.) Blocks, which are3-60 amino acids in length, correspond to the most highly conservedregions of proteins. The BLOCKS algorithm compares a query sequence witha weighted scoring matrix of blocks in the BLOCKS database. Blocks inthe BLOCKS database are calibrated against protein sequences with knownfunctions from the SWISS-PROT database to determine the stochasticdistribution of matches. Similar databases such as PRINTS, a proteinfingerprint database, are also searchable using the BLOCKS algorithm.(Attwood, T. K. et al. (1997) J. Chem. Inf. Comput. Sci. 37:417-424.)PRINTS is based on non-redundant sequences obtained from sources such asSWISS-PROT, GenBank, PIR, and NRL-3D.

[0188] The BLOCKS algorithm searches for matches between a querysequence and the BLOCKS or PRINTS database and evaluates the statisticalsignificance of any matches found. Matches from a BLOCKS or PRINTSsearch can be evaluated on two levels, local similarity and globalsimilarity. The degree of local similarity is measured by scores, andthe extent of global similarity is measured by score ranking andprobability values. A score of 1000 or greater for a BLOCKS match ofhighest ranking indicates that the match falls within the 0.5 percentilelevel of false positives when the matched block is calibrated againstSWISS-PROT. Likewise, a probability value of less than 1.0×10⁻³indicates that the match would occur by chance no more than one time inevery 1000 searches. Only those matches with a cutoff score of 1000 orgreater and a cutoff probability value of 1.0×10⁻³ or less areconsidered in the functional analyses of the protein sequences in theSequence Listing.

[0189] Nucleic and amino acid sequences of the Sequence Listing may alsobe analyzed using PFAM. PFAM is a Hidden Markov Model (HMM) basedprotocol useful in protein family searching. HMM is a probabilisticapproach which analyzes consensus primary structures of gene families.(See, e.g., Eddy, S. R. (1996) Cur. Opin. Str. Biol. 6:361-365.)

[0190] The PFAM database contains protein sequences of 527 proteinfamilies gathered from publicly available sources, e.g., SWISS-PROT andPROSITE. PFAM searches for well characterized protein domain familiesusing two high-quality alignment routines, seed alignment and fullalignment. (See, e.g., Sonnhammer, E. L. L. et al. (1997) Proteins28:405-420.) The seed alignment utilizes the hmmls program, a programthat searches for local matches, and a non-redundant set of the PFAMdatabase. The full alignment utilizes the hmmfs program, a program thatsearches for multiple fragments in long sequences, e.g., repeats andmotifs, and all sequences in the PFAM database. A result or score of 100“bits” can signify that it is 2¹⁰⁰-fold more likely that the sequence isa true match to the model or comparison sequence. Cutoff scores whichrange from 10 to 50 bits are generally used for individual proteinfamilies using the SWISS-PROT sequences as model or comparisonsequences.

[0191] Two other algorithms, SIGPEPT and TM, both based on the HMMalgorithm described above (see, e.g., Eddy, supra; and Sonnhammer,supra), identify potential signal sequences and transmembrane domains,respectively. SIGPEPT was created using protein sequences having signalsequence annotations derived from SWISS-PROT. It contains about 1413non-redundant signal sequences ranging in length from 14 to 36 aminoacid residues. TM was created similarly using transmembrane domainannotations. It contains about 453 non-redundant transmembrane sequencesencompassing 1579 transmembrane domain segments. Suitable HMM modelswere constructed using the above sequences and were refined with knownSWISS-PROT signal peptide sequences or transmembrane domain sequencesuntil a high correlation coefficient, a measurement of the correctnessof the analysis, was obtained. Using the protein sequences from theSWISS-PROT database as a test set, a cutoff score of 11 bits, asdetermined above, correlated with 91-94% true-positives and about 4.1%false-positives, yielding a correlation coefficient of about 0.87-0.90for SIGPEPT. A score of 11 bits for TM will typically give the followingresults: 75% true positives; 1.72% false positives; and a correlationcoefficient of 0.76. Each search evaluates the statistical significanceof any matches found and reports only those matches that score at least11 bits.

[0192] IV. Northern Analysis

[0193] Northern analysis is a laboratory technique used to detect thepresence of a transcript of a gene and involves the hybridization of alabeled nucleotide sequence to a membrane on which RNAs from aparticular cell type or tissue have been bound. (See, e.g., Sambrook,supra, ch. 7; and Ausubel, supra, ch. 4 and 16.)

[0194] Analogous computer techniques applying BLAST are used to searchfor identical or related molecules in nucleotide databases such asGenBank or the LIFESEQ database (Incyte Pharmaceuticals). This analysisis much faster than multiple membrane-based hybridizations. In addition,the sensitivity of the computer search can be modified to determinewhether any particular match is categorized as exact or similar.

[0195] The basis of the search is the product score, which is definedas:

% sequence identity×% maximum BLAST score/100

[0196] The product score takes into account both the degree ofsimilarity between two sequences and the length of the sequence match.For example, with a product score of 40, the match will be exact withina 1% to 2% error, and, with a product score of 70, the match will beexact. Similar molecules are usually identified by selecting those whichshow product scores between 15 and 40, although lower scores mayidentify related molecules.

[0197] The results of Northern analysis are reported as a list oflibraries in which the transcript encoding DRPCS occurs. Abundance andpercent abundance are also reported. Abundance directly reflects thenumber of times a particular transcript is represented in a cDNAlibrary, and percent abundance is abundance divided by the total numberof sequences examined in the cDNA library.

[0198] V. Extension of DRPCS Encoding Polynucleotides

[0199] The nucleic acid sequence of Incyte Clone 637471 was used todesign oligonucleotide primers for extending a partial nucleotidesequence to full length. One primer was synthesized to initiateextension of an antisense polynucleotide, and the other was synthesizedto initiate extension of a sense polynucleotide. Primers were used tofacilitate the extension of the known sequence “outward” generatingamplicons containing new unknown nucleotide sequence for the region ofinterest. The initial primers were designed from the cDNA using OLIGO4.06 software (National Biosciences, Plymouth, Minn.), or anotherappropriate program, to be about 22 to 30 nucleotides in length, to havea GC content of about 50% or more, and to anneal to the target sequenceat temperatures of about 68° C. to about 72° C. Any stretch ofnucleotides which would result in hairpin structures and primer-primerdimerizations was avoided.

[0200] Selected human cDNA libraries (GIBCO BRL) were used to extend thesequence. If more than one extension is necessary or desired, additionalsets of primers are designed to further extend the known region.

[0201] High fidelity amplification was obtained by following theinstructions for the XL-PCR kit (Perkin Elmer) and thoroughly mixing theenzyme and reaction mix. PCR was performed using the PTC200 thermalcycler (M. J. Research, Watertown, Mass.), beginning with 40 pmol ofeach primer and the recommended concentrations of all other componentsof the kit, with the following parameters: Step 1 94° C. for 1 min(initial denaturation) Step 2 65° C. for 1 min Step 3 68° C. for 6 minStep 4 94° C. for 15 sec Step 5 65° C. for 1 min Step 6 68° C. for 7 minStep 7 Repeat steps 4 through 6 for an additional 15 cycles Step 8 94°C. for 15 sec Step 9 65° C. for 1 min Step 10 68° C. for 7:15 min Step11 Repeat steps 8 through 10 for an additional 12 cycles Step 12 72° C.for 8 min Step 13  4° C. (and holding)

[0202] A 5 μl to 10 μl aliquot of the reaction mixture was analyzed byelectrophoresis on a low concentration (about 0.6% to 0.8%) agarosemini-gel to determine which reactions were successful in extending thesequence. Bands thought to contain the largest products were excisedfrom the gel, purified using the QIAQUICK purification system (QIAGENInc.), and trimmed of overhangs using Klenow enzyme to facilitatereligation and cloning.

[0203] After ethanol precipitation, the products were redissolved in 13μl of ligation buffer, 1 μl T4-DNA ligase (15 units) and 1 μT4polynucleotide kinase were added, and the mixture was incubated at roomtemperature for 2 to 3 hours, or overnight at 16° C. Competent E. colicells (in 40 μl of appropriate media) were transformed with 3 μl ofligation mixture and cultured in 80 μl of SOC medium. (See, e.g.,Sambrook, supra, Appendix A, p. 2.) After incubation for one hour at 37°C., the E. coli mixture was plated on Luria Bertani (LB) agar (See,e.g., Sambrook, supra, Appendix A, p. 1) containing carbenicillin(2×carb). The following day, several colonies were randomly picked fromeach plate and cultured in 150 μl of liquid LB/2×carb medium placed inan individual well of an appropriate commercially-available sterile96-well microtiter plate. The following day, 5 μl of each overnightculture was transferred into a non-sterile 96-well plate and, afterdilution 1:10 with water, 5 μl from each sample was transferred into aPCR array.

[0204] For PCR amplification, 18 μl of concentrated PCR reaction mix(3.3×) containing 4 units of rTth DNA polymerase, a vector primer, andone or both of the gene specific primers used for the extension reactionwere added to each well. Amplification was performed using the followingconditions: Step 1 94° C. for 60 sec Step 2 94° C. for 20 sec Step 3 55°C. for 30 sec Step 4 72° C. for 90 sec Step 5 Repeat steps 2 through 4for an additional 29 cycles Step 6 72° C. for 180 sec Step 7  4° C. (andholding)

[0205] Aliquots of the PCR reactions were run on agarose gels togetherwith molecular weight markers. The sizes of the PCR products werecompared to the original partial cDNAs, and appropriate clones wereselected, ligated into plasmid, and sequenced.

[0206] In like manner, the nucleotide sequence of SEQ ID NO:2 is used toobtain 5′ regulatory sequences using the procedure above,oligonucleotides designed for 5′ extension, and an appropriate genomiclibrary.

[0207] VI. Labeling and Use of Individual Hybridization Probes

[0208] Hybridization probes derived from SEQ ID NO:2 are employed toscreen cDNAs, genomic DNAs, or mRNAs. Although the labeling ofoligonucleotides, consisting of about 20 base pairs, is specificallydescribed, essentially the same procedure is used with larger nucleotidefragments. Oligonucleotides are designed using state-of-the-art softwaresuch as OLIGO 4.06 software (National Biosciences) and labeled bycombining 50 pmol of each oligomer, 250 μCi of [γ-³²P] adenosinetriphosphate (Amersham, Chicago, Ill.), and T4 polynucleotide kinase(DuPont NEN, Boston, Mass.). The labeled oligonucleotides aresubstantially purified using a SEPHADEX G-25 superfine size exclusiondextran bead column (Pharmacia & Upjohn, Kalamazoo, Mich.). An aliquotcontaining 10⁷ counts per minute of the labeled probe is used in atypical membrane-based hybridization analysis of human genomic DNAdigested with one of the following endonucleases: Ase I, Bgl II, Eco RI,Pst I, Xbal, or Pvu II (DuPont NEN, Boston, Mass.).

[0209] The DNA from each digest is fractionated on a 0.7% agarose geland transferred to nylon membranes (Nytran Plus, Schleicher & Schuell,Durham, N.H.). Hybridization is carried out for 16 hours at 40° C. Toremove nonspecific signals, blots are sequentially washed at roomtemperature under increasingly stringent conditions up to 0.1×salinesodium citrate and 0.5% sodium dodecyl sulfate. After XOMAT AR film(Kodak, Rochester, N.Y.) is exposed to the blots, hybridization patternsare compared visually.

[0210] VII. Microarrays

[0211] A chemical coupling procedure and an ink jet device can be usedto synthesize array elements on the surface of a substrate. (See, e.g.,Baldeschweiler, supra.) An array analogous to a dot or slot blot mayalso be used to arrange and link elements to the surface of a substrateusing thermal, UV, chemical, or mechanical bonding procedures. A typicalarray may be produced by hand or using available methods and machinesand contain any appropriate number of elements. After hybridization,nonhybridized probes are removed and a scanner used to determine thelevels and patterns of fluorescence. The degree of complementarity andthe relative abundance of each probe which hybridizes to an element onthe microarray may be assessed through analysis of the scanned images.

[0212] Full-length cDNAs, Expressed Sequence Tags (ESTs), or fragmentsthereof may comprise the elements of the microarray. Fragments suitablefor hybridization can be selected using software well known in the artsuch as LASERGENE software (DNASTAR Inc). Full-length cDNAs, ESTs, orfragments thereof corresponding to one of the nucleotide sequences ofthe present invention, or selected at random from a cDNA libraryrelevant to the present invention, are arranged on an appropriatesubstrate, e.g., a glass slide. The cDNA is fixed to the slide using,e.g., UV cross-linking followed by thermal and chemical treatments andsubsequent drying. (See, e.g., Schena, M. et al. (1995) Science270:467-470; and Shalon, D. et al. (1996) Genome Res. 6:639-645.)Fluorescent probes are prepared and used for hybridization to theelements on the substrate. The substrate is analyzed by proceduresdescribed above.

[0213] VIII. Complementary Polynucleotides

[0214] Sequences complementary to the DRPCS-encoding sequences, or anyparts thereof, are used to detect, decrease, or inhibit expression ofnaturally occurring DRPCS. Although use of oligonucleotides comprisingfrom about 15 to 30 base pairs is described, essentially the sameprocedure is used with smaller or with larger sequence fragments.Appropriate oligonucleotides are designed using OLIGO 4.06 software andthe coding sequence of DRPCS. To inhibit transcription, a complementaryoligonucleotide is designed from the most unique 5′ sequence and used toprevent promoter binding to the coding sequence. To inhibit translation,a complementary oligonucleotide is designed to prevent ribosomal bindingto the DRPCS-encoding transcript.

[0215] IX. Expression of DRPCS

[0216] Expression and purification of DRPCS is achieved using bacterialor virus-based expression systems. For expression of DRPCS in bacteria,cDNA is subcloned into an appropriate vector containing an antibioticresistance gene and an inducible promoter that directs high levels ofcDNA transcription. Examples of such promoters include, but are notlimited to, the trp-lac (tac) hybrid promoter and the T5 or T7bacteriophage promoter in conjunction with the lac operator regulatoryelement. Recombinant vectors are transformed into suitable bacterialhosts, e.g., BL21(DE3). Antibiotic resistant bacteria express DRPCS uponinduction with isopropyl beta-D-thiogalactopyranoside (IPTG). Expressionof DRPCS in eukaryotic cells is achieved by infecting insect ormammalian cell lines with recombinant Autographica califonica nuclearpolyhedrosis virus (AcMNPV), commonly known as baculovirus. Thenonessential polyhedrin gene of baculovirus is replaced with cDNAencoding DRPCS by either homologous recombination or bacterial-mediatedtransposition involving transfer plasmid intermediates. Viralinfectivity is maintained and the strong polyhedrin promoter drives highlevels of cDNA transcription. Recombinant baculovirus is used to infectSpodoptera frugiperda (Sf9) insect cells in most cases, or humanhepatocytes, in some cases. Infection of the latter requires additionalgenetic modifications to baculovirus. (See Engelhard, E. K. et al.(1994) Proc. Natl. Acad. Sci. USA 91:3224-3227; Sandig, V. et al. (1996)Hum. Gene Ther. 7:1937-1945.)

[0217] In most expression systems, DRPCS is synthesized as a fusionprotein with, e.g., glutathione S-transferase (GST) or a peptide epitopetag, such as FLAG or 6-His, permitting rapid, single-step,affinity-based purification of recombinant fusion protein from crudecell lysates. GST, a 26-kilodalton enzyme from Schistosomajaponicum,enables the purification of fusion proteins on immobilized glutathioneunder conditions that maintain protein activity and antigenicity(Pharmacia, Piscataway, N.J.). Following purification, the GST moietycan be proteolytically cleaved from DRPCS at specifically engineeredsites. FLAG, an 8-amino acid peptide, enables immunoaffinitypurification using commercially available monoclonal and polyclonalanti-FLAG antibodies (Eastman Kodak, Rochester, N.Y.). 6-His, a stretchof six consecutive histidine residues, enables purification onmetal-chelate resins (QIAGEN Inc, Chatsworth, Calif.). Methods forprotein expression and purification are discussed in Ausubel, F. M. etal. (1995 and periodic supplements) Current Protocols in MolecularBiology, John Wiley & Sons, New York, N.Y., ch 10, 16. Purified DRPCSobtained by these methods can be used directly in the following activityassay.

[0218] X. Demonstration of DRPCS Activity

[0219] Activity of DRPCS is measured by determining the potassiumcurrent using single-cell patch-clamp analysis. Cells expressing DRPCSare dissociated from one another, and single cells are selected.Potassium currents are measured with a two-microelectrode voltage clamp.The standard extracellular recording solution is 80 mM NaCl, 5 mM KCl,1.8 mM CaCl₂, 1 mM MgCl₂, and 5 mM Na-HEPES, pH 7.6. The intracellularelectrode is filled with 3 M KCl with a resistance of 0.5-3 MΩ.Stimulation, sampling and data collection are performed by computer. Thepotassium channel blocking agent, tetraethylammonium, definesnon-specific, or leak, potassium currents. Following a depolarizingpulse, the characteristics of the resulting potassium current aremeasured via the recording electrodes. The amount of potassium currentthat flows in response to a unit depolarization is proportional to theactivity of DRPCS in the cell. (Shi, W. et al. (1997) J. Neurosci.17:9423-9432; Stühmer, W. et al. (1989) EMBO J. 8:3235-3244.)

[0220] XI. Functional Assays

[0221] DRPCS function is assessed by expressing the sequences encodingDRPCS at physiologically elevated levels in mammalian cell culturesystems. cDNA is subcloned into a mammalian expression vector containinga strong promoter that drives high levels of cDNA expression. Vectors ofchoice include PCMV SPORT plasmid (Life Technologies, Gaithersburg, Md.)and PCR3.1 plasmid (Invitrogen, Carlsbad, Calif., both of which containthe cytomegalovirus promoter. 5-10 μg of recombinant vector aretransiently transfected into a human cell line, preferably ofendothelial or hematopoietic origin, using either liposome formulationsor electroporation. 1-2 μg of an additional plasmid containing sequencesencoding a marker protein are co-transfected. Expression of a markerprotein provides a means to distinguish transfected cells fromnontransfected cells and is a reliable predictor of cDNA expression fromthe recombinant vector. Marker proteins of choice include, e.g., GreenFluorescent Protein (GFP) (Clontech, Palo Alto, Calif.), CD64, or aCD64-GFP fusion protein. Flow cytometry (FCM), an automated, laseroptics-based technique, is used to identify transfected cells expressingGFP or CD64-GFP, and to evaluate properties, for example, theirapoptotic state. FCM detects and quantifies the uptake of fluorescentmolecules that diagnose events preceding or coincident with cell death.These events include changes in nuclear DNA content as measured bystaining of DNA with propidium iodide; changes in cell size andgranularity as measured by forward light scatter and 90 degree sidelight scatter; down-regulation of DNA synthesis as measured by decreasein bromodeoxyuridine uptake; alterations in expression of cell surfaceand intracellular proteins as measured by reactivity with specificantibodies; and alterations in plasma membrane composition as measuredby the binding of fluorescein-conjugated Annexin V protein to the cellsurface. Methods in flow cytometry are discussed in Ormerod, M. G.(1994) Flow Cytometry, Oxford, New York, N.Y.

[0222] The influence of DRPCS on gene expression can be assessed usinghighly purified populations of cells transfected with sequences encodingDRPCS and either CD64 or CD64-GFP. CD64 and CD64-GFP are expressed onthe surface of transfected cells and bind to conserved regions of humanimmunoglobulin G (IgG). Transfected cells are efficiently separated fromnontransfected cells using magnetic beads coated with either human IgGor antibody against CD64 (DYNAL, Lake Success, N.Y.). mRNA can bepurified from the cells using methods well known by those of skill inthe art. Expression of mRNA encoding DRPCS and other genes of interestcan be analyzed by Northern analysis or microarray techniques.

[0223] XII. Production of DRPCS Specific Antibodies

[0224] DRPCS substantially purified using polyacrylamide gelelectrophoresis (PAGE)(see, e.g., Harrington, M. G. (1990) MethodsEnzymol. 182:488-495), or other purification techniques, is used toimmunize rabbits and to produce antibodies using standard protocols.

[0225] Alternatively, the DRPCS amino acid sequence is analyzed usingLASERGENE software (DNASTAR Inc.) to determine regions of highimmunogenicity, and a corresponding oligopeptide is synthesized and usedto raise antibodies by means known to those of skill in the art. Methodsfor selection of appropriate epitopes, such as those near the C-terminusor in hydrophilic regions are well described in the art. (See, e.g.,Ausubel supra, ch. 11.)

[0226] Typically, oligopeptides 15 residues in length are synthesizedusing an ABI 431A peptide synthesizer (Applied Biosystems) usingfmoc-chemistry and coupled to KLH (Sigma, St. Louis, Mo.) by reactionwith N-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) to increaseimmunogenicity. (See, e.g., Ausubel supra.) Rabbits are immunized withthe oligopeptide-KLH complex in complete Freund's adjuvant. Resultingantisera are tested for antipeptide activity by, for example, bindingthe peptide to plastic, blocking with 1% BSA, reacting with rabbitantisera, washing, and reacting with radio-iodinated goat anti-rabbitIgG.

[0227] XIII. Purification of Naturally Occurring DRPCS Using SpecificAntibodies

[0228] Naturally occurring or recombinant DRPCS is substantiallypurified by immunoaffinity chromatography using antibodies specific forDRPCS. An immunoaffinity column is constructed by covalently couplinganti-DRPCH antibody to an activated chromatographic resin, such asCNBr-activated SEPHAROSE (Pharmacia & Upjohn). After the coupling, theresin is blocked and washed according to the manufacturer'sinstructions.

[0229] Media containing DRPCS are passed over the immunoaffinity column,and the column is washed under conditions that allow the preferentialabsorbance of DRPCS (e.g., high ionic strength buffers in the presenceof detergent). The column is eluted under conditions that disruptantibody/DRPCH binding (e.g., a buffer of pH 2 to pH 3, or a highconcentration of a chaotrope, such as urea or thiocyanate ion), andDRPCS is collected.

[0230] XIV. Identification of Molecules Which Interact with DRPCS

[0231] DRPCS, or biologically active fragments thereof, are labeled with¹²⁵I Bolton-Hunter reagent. (See, e.g., Bolton et al. (1973) Biochem. J.133:529.) Candidate molecules previously arrayed in the wells of amulti-well plate are incubated with the labeled DRPCS, washed, and anywells with labeled DRPCS complex are assayed. Data obtained usingdifferent concentrations of DRPCS are used to calculate values for thenumber, affinity, and association of DRPCS with the candidate molecules.

[0232] Various modifications and variations of the described methods andsystems of the invention will be apparent to those skilled in the artwithout departing from the scope and spirit of the invention. Althoughthe invention has been described in connection with specific preferredembodiments, it should be understood that the invention as claimedshould not be unduly limited to such specific embodiments. Indeed,various modifications of the described modes for carrying out theinvention which are obvious to those skilled in molecular biology orrelated fields are intended to be within the scope of the followingclaims.

1 4 170 amino acids amino acid single linear BRSTNOT03 637471 1 Met LeuLys Met Glu Pro Leu Asn Ser Thr His Pro Gly Thr Ala Ala 1 5 10 15 SerSer Ser Pro Leu Glu Ser Arg Ala Ala Gly Gly Gly Ser Gly Asn 20 25 30 GlyAsn Glu Tyr Phe Tyr Ile Leu Val Val Met Ser Phe Tyr Gly Ile 35 40 45 PheLeu Ile Gly Ile Met Leu Gly Tyr Met Lys Ser Lys Arg Arg Glu 50 55 60 LysLys Ser Ser Leu Leu Leu Leu Tyr Lys Asp Glu Glu Arg Leu Trp 65 70 75 80Gly Glu Ala Met Lys Pro Leu Pro Val Val Ser Gly Leu Arg Ser Val 85 90 95Gln Val Pro Leu Met Leu Asn Met Leu Gln Glu Ser Val Ala Pro Ala 100 105110 Leu Ser Cys Thr Leu Cys Ser Met Glu Gly Asp Ser Val Ser Ser Glu 115120 125 Ser Ser Ser Pro Asp Val His Leu Thr Ile Gln Glu Glu Gly Ala Asp130 135 140 Glu Glu Leu Glu Glu Thr Ser Glu Thr Pro Leu Asn Glu Ser SerGlu 145 150 155 160 Gly Ser Ser Glu Asn Ile His Gln Asn Ser 165 170 645base pairs nucleic acid single linear BRSTNOT03 637471 2 CAGAGCAGAAGAACCCTCTT GGACTGGACG ATTTGGGAAT TCAAAACTTG GGACAAACTG 60 TCAGCCTTGCCCCTGCTGTG GAGGCAGCCT CAATGCTGAA AATGGAGCCT CTGAACAGCA 120 CGCACCCCGGCACCGCCGCC TCCAGCAGCC CCCTGGAGTC CCGTGCGGCC GGTGGCGGCA 180 GCGGCAATGGCAACGAGTAC TTCTACATTC TGGTTGTCAT GTCCTTCTAC GGCATTTTCT 240 TGATCGGAATCATGCTGGGC TACATGAAAT CCAAGAGGCG GGAGAAGAAG TCCAGCCTCC 300 TGCTGCTGTACAAAGACGAG GAGCGGCTCT GGGGGGAGGC CATGAAGCCG CTGCCCGTGG 360 TGTCGGGCCTGAGGTCGGTG CAGGTGCCCC TGATGCTGAA CATGCTGCAG GAGAGCGTGG 420 CGCCCGCGCTGTCCTGCACC CTCTGTTCCA TGGAAGGGGA CAGCGTGAGC TCCGAGTCCT 480 CCTCCCCGGACGTGCACCTC ACCATTCAGG AGGAGGGGGC AGACGAGGAG CTGGAGGAGA 540 CCTCGGAGACGCCCCTCAAC GAGAGCAGCG AAGGGTCCTC GGAGAACATC CATCAGAATT 600 CCTAGCANCCCCGGGAACCC TGCGGGTGGC TCCCATCAGC AGCAA 645 129 amino acids amino acidsingle linear GenBank 452497 3 Met Ile Leu Ser Asn Thr Thr Ala Val ThrPro Phe Leu Thr Lys Leu 1 5 10 15 Trp Gln Glu Thr Val Gln Gln Gly GlyAsn Met Ser Gly Leu Ala Arg 20 25 30 Arg Ser Pro Arg Ser Gly Asp Gly LysLeu Glu Ala Leu Tyr Val Leu 35 40 45 Met Val Leu Gly Phe Phe Gly Phe PheThr Leu Gly Ile Met Leu Ser 50 55 60 Tyr Ile Arg Ser Lys Lys Leu Glu HisSer Asn Asp Pro Phe Asn Val 65 70 75 80 Tyr Ile Glu Ser Asp Ala Trp GlnGlu Lys Asp Lys Ala Tyr Val Gln 85 90 95 Ala Arg Val Leu Glu Ser Tyr ArgSer Cys Tyr Val Val Glu Asn His 100 105 110 Leu Ala Ile Glu Gln Pro AsnThr His Leu Pro Glu Thr Lys Pro Ser 115 120 125 Pro 130 amino acidsamino acid single linear GenBank 203977 4 Met Ala Leu Ser Asn Ser ThrThr Val Leu Pro Phe Leu Ala Ser Leu 1 5 10 15 Trp Gln Glu Thr Asp GluPro Gly Gly Asn Met Ser Ala Asp Leu Ala 20 25 30 Arg Arg Ser Gln Leu ArgAsp Asp Ser Lys Leu Glu Ala Leu Tyr Ile 35 40 45 Leu Met Val Leu Gly PhePhe Gly Phe Phe Thr Leu Gly Ile Met Leu 50 55 60 Ser Tyr Ile Arg Ser LysLys Leu Glu His Ser His Asp Pro Phe Asn 65 70 75 80 Val Tyr Ile Glu SerAsp Ala Trp Gln Glu Lys Gly Lys Ala Leu Phe 85 90 95 Gln Ala Arg Val LeuGlu Ser Phe Arg Ala Cys Tyr Val Ile Glu Asn 100 105 110 Gln Ala Ala ValGlu Gln Pro Ala Thr His Leu Pro Glu Leu Lys Pro 115 120 125 Leu Ser 130

What is claimed is:
 1. An isolated polypeptide selected from the groupconsisting of: a) a polypeptide comprising the amino acid sequence ofSEQ ID NO:1, b) a polypeptide comprising a naturally occurring aminoacid sequence at least 90% identical to the amino acid sequence of SEQID NO:1, c) a biologically active fragment of a polypeptide having theamino acid sequence of SEQ ID NO:1, and d) an immunogenic fragment of apolypeptide having the amino acid sequence of SEQ ID NO:1.
 2. Anisolated polypeptide of claim 1 comprising the amino acid sequence ofSEQ ID NO:1.
 3. An isolated polynucleotide encoding a polypeptide ofclaim
 1. 4. An isolated polynucleotide encoding a polypeptide of claim2.
 5. An isolated polynucleotide of claim 4 comprising thepolynucleotide sequence of SEQ ID NO:2.
 6. A recombinant polynucleotidecomprising a promoter sequence operably linked to a polynucleotide ofclaim
 3. 7. A cell transformed with a recombinant polynucleotide ofclaim
 6. 8. A transgenic organism comprising a recombinantpolynucleotide of claim
 6. 9. A method of producing a polypeptide ofclaim 1, the method comprising: a) culturing a cell under conditionssuitable for expression of the polypeptide, wherein said cell istransformed with a recombinant polynucleotide, and said recombinantpolynucleotide comprises a promoter sequence operably linked to apolynucleotide encoding the polypeptide of claim 1, and b) recoveringthe polypeptide so expressed.
 10. A method of claim 9, wherein thepolypeptide comprises the amino acid sequence of SEQ ID NO:1.
 11. Anisolated antibody which specifically binds to a polypeptide of claim 1.12. An isolated polynucleotide selected from the group consisting of: a)a polynucleotide comprising the polynucleotide sequence of SEQ ID NO:2,b) a polynucleotide comprising a naturally occurring polynucleotidesequence at least 90% identical to the polynucleotide sequence of SEQ IDNO:2, c) a polynucleotide complementary to a polynucleotide of a), d) apolynucleotide complementary to a polynucleotide of b), and e) an RNAequivalent of a)-d).
 13. An isolated polynucleotide comprising at least60 contiguous nucleotides of a polynucleotide of claim
 12. 14. A methodof detecting a target polynucleotide in a sample, said targetpolynucleotide having a sequence of a polynucleotide of claim 12, themethod comprising: a) hybridizing the sample with a probe comprising atleast 20 contiguous nucleotides comprising a sequence complementary tosaid target polynucleotide in the sample, and which probe specificallyhybridizes to said target polynucleotide, under conditions whereby ahybridization complex is formed between said probe and said targetpolynucleotide or fragments thereof, and b) detecting the presence orabsence of said hybridization complex, and, optionally, if present, theamount thereof.
 15. A method of claim 14, wherein the probe comprises atleast 60 contiguous nucleotides.
 16. A method of detecting a targetpolynucleotide in a sample, said target polynucleotide having a sequenceof a polynucleotide of claim 12, the method comprising: a) amplifyingsaid target polynucleotide or fragment thereof using polymerase chainreaction amplification, and b) detecting the presence or absence of saidamplified target polynucleotide or fragment thereof, and, optionally, ifpresent, the amount thereof.
 17. A composition comprising a polypeptideof claim 1 and a pharmaceutically acceptable excipient.
 18. Acomposition of claim 17, wherein the polypeptide comprises the aminoacid sequence of SEQ ID NO:1.
 19. A method for treating a disease orcondition associated with decreased expression of functional DRPCS,comprising administering to a patient in need of such treatment thecomposition of claim
 17. 20. A method of screening a compound foreffectiveness as an agonist of a polypeptide of claim 1, the methodcomprising: a) exposing a sample comprising a polypeptide of claim 1 toa compound, and b) detecting agonist activity in the sample.
 21. Acomposition comprising an agonist compound identified by a method ofclaim 20 and a pharmaceutically acceptable excipient.
 22. A method fortreating a disease or condition associated with decreased expression offunctional DRPCS, comprising administering to a patient in need of suchtreatment a composition of claim
 21. 23. A method of screening acompound for effectiveness as an antagonist of a polypeptide of claim 1,the method comprising: a) exposing a sample comprising a polypeptide ofclaim 1 to a compound, and b) detecting antagonist activity in thesample.
 24. A composition comprising an antagonist compound identifiedby a method of claim 23 and a pharmaceutically acceptable excipient. 25.A method for treating a disease or condition associated withoverexpression of functional DRPCS, comprising administering to apatient in need of such treatment a composition of claim
 24. 26. Amethod of screening for a compound that specifically binds to thepolypeptide of claim 1, the method comprising: a) combining thepolypeptide of claim 1 with at least one test compound under suitableconditions, and b) detecting binding of the polypeptide of claim 1 tothe test compound, thereby identifying a compound that specificallybinds to the polypeptide of claim
 1. 27. A method of screening for acompound that modulates the activity of the polypeptide of claim 1, themethod comprising: a) combining the polypeptide of claim 1 with at leastone test compound under conditions permissive for the activity of thepolypeptide of claim 1, b) assessing the activity of the polypeptide ofclaim 1 in the presence of the test compound, and c) comparing theactivity of the polypeptide of claim 1 in the presence of the testcompound with the activity of the polypeptide of claim 1 in the absenceof the test compound, wherein a change in the activity of thepolypeptide of claim 1 in the presence of the test compound isindicative of a compound that modulates the activity of the polypeptideof claim
 1. 28. A method of screening a compound for effectiveness inaltering expression of a target polynucleotide, wherein said targetpolynucleotide comprises a sequence of claim 5, the method comprising:a) exposing a sample comprising the target polynucleotide to a compound,under conditions suitable for the expression of the targetpolynucleotide, b) detecting altered expression of the targetpolynucleotide, and c) comparing the expression of the targetpolynucleotide in the presence of varying amounts of the compound and inthe absence of the compound.
 29. A method of assessing toxicity of atest compound, the method comprising: a) treating a biological samplecontaining nucleic acids with the test compound, b) hybridizing thenucleic acids of the treated biological sample with a probe comprisingat least 20 contiguous nucleotides of a polynucleotide of claim 12 underconditions whereby a specific hybridization complex is formed betweensaid probe and a target polynucleotide in the biological sample, saidtarget polynucleotide comprising a polynucleotide sequence of apolynucleotide of claim 12 or fragment thereof, c) quantifying theamount of hybridization complex, and d) comparing the amount ofhybridization complex in the treated biological sample with the amountof hybridization complex in an untreated biological sample, wherein adifference in the amount of hybridization complex in the treatedbiological sample is indicative of toxicity of the test compound.
 30. Adiagnostic test for a condition or disease associated with theexpression of DRPCS in a biological sample, the method comprising: a)combining the biological sample with an antibody of claim 11, underconditions suitable for the antibody to bind the polypeptide and form anantibody:polypeptide complex, and b) detecting the complex, wherein thepresence of the complex correlates with the presence of the polypeptidein the biological sample.
 31. The antibody of claim 11, wherein theantibody is: a) a chimeric antibody, b) a single chain antibody, c) aFab fragment, d) a F(ab′)₂ fragment, or e) a humanized antibody.
 32. Acomposition comprising an antibody of claim 11 and an acceptableexcipient.
 33. A method of diagnosing a condition or disease associatedwith the expression of DRPCS in a subject, comprising administering tosaid subject an effective amount of the composition of claim
 32. 34. Acomposition of claim 32, wherein the antibody is labeled.
 35. A methodof diagnosing a condition or disease associated with the expression ofDRPCS in a subject, comprising administering to said subject aneffective amount of the composition of claim
 34. 36. A method ofpreparing a polyclonal antibody with the specificity of the antibody ofclaim 11, the method comprising: a) immunizing an animal with apolypeptide consisting of the amino acid sequence of SEQ ID NO:1, or animmunogenic fragment thereof, under conditions to elicit an antibodyresponse, b) isolating antibodies from said animal, and c) screening theisolated antibodies with the polypeptide, thereby identifying apolyclonal antibody which specifically binds to a polypeptide comprisingthe amino acid sequence of SEQ ID NO:1.
 37. A polyclonal antibodyproduced by a method of claim
 36. 38. A composition comprising thepolyclonal antibody of claim 37 and a suitable carrier.
 39. A method ofmaking a monoclonal antibody with the specificity of the antibody ofclaim 11, the method comprising: a) immunizing an animal with apolypeptide consisting of the amino acid sequence of SEQ ID NO:1, or animmunogenic fragment thereof, under conditions to elicit an antibodyresponse, b) isolating antibody producing cells from the animal, c)fusing the antibody producing cells with immortalized cells to formmonoclonal antibody-producing hybridoma cells, d) culturing thehybridoma cells, and e) isolating from the culture monoclonal antibodywhich specifically binds to a polypeptide comprising the amino acidsequence of SEQ ID NO:1.
 40. A monoclonal antibody produced by a methodof claim
 39. 41. A composition comprising the monoclonal antibody ofclaim 40 and a suitable carrier.
 42. The antibody of claim 11, whereinthe antibody is produced by screening a Fab expression library.
 43. Theantibody of claim 11, wherein the antibody is produced by screening arecombinant immunoglobulin library.
 44. A method of detecting apolypeptide comprising the amino acid sequence of SEQ I) NO:1 in asample, the method comprising: a) incubating the antibody of claim 11with a sample under conditions to allow specific binding of the antibodyand the polypeptide, and b) detecting specific binding, wherein specificbinding indicates the presence of a polypeptide comprising the aminoacid sequence of SEQ ID NO:1 in the sample.
 45. A method of purifying apolypeptide comprising the amino acid sequence of SEQ ID NO:1 from asample, the method comprising: a) incubating the antibody of claim 11with a sample under conditions to allow specific binding of the antibodyand the polypeptide, and b) separating the antibody from the sample andobtaining the purified polypeptide comprising the amino acid sequence ofSEQ ID NO:1.
 46. A microarray wherein at least one element of themicroarray is a polynucleotide of claim
 13. 47. A method of generatingan expression profile of a sample which contains polynucleotides, themethod comprising: a) labeling the polynucleotides of the sample, b)contacting the elements of the microarray of claim 46 with the labeledpolynucleotides of the sample under conditions suitable for theformation of a hybridization complex, and c) quantifying the expressionof the polynucleotides in the sample.
 48. An array comprising differentnucleotide molecules affixed in distinct physical locations on a solidsubstrate, wherein at least one of said nucleotide molecules comprises afirst oligonucleotide or polynucleotide sequence specificallyhybridizable with at least 30 contiguous nucleotides of a targetpolynucleotide, and wherein said target polynucleotide is apolynucleotide of claim
 12. 49. An array of claim 48, wherein said firstoligonucleotide or polynucleotide sequence is completely complementaryto at least 30 contiguous nucleotides of said target polynucleotide. 50.An array of claim 48, wherein said first oligonucleotide orpolynucleotide sequence is completely complementary to at least 60contiguous nucleotides of said target polynucleotide.
 51. An array ofclaim 48, wherein said first oligonucleotide or polynucleotide sequenceis completely complementary to said target polynucleotide.
 52. An arrayof claim 48, which is a microarray.
 53. An array of claim 48, furthercomprising said target polynucleotide hybridized to a nucleotidemolecule comprising said first oligonucleotide or polynucleotidesequence.
 54. An array of claim 48, wherein a linker joins at least oneof said nucleotide molecules to said solid substrate.
 55. An array ofclaim 48, wherein each distinct physical location on the substratecontains multiple nucleotide molecules, and the multiple nucleotidemolecules at any single distinct physical location have the samesequence, and each distinct physical location on the substrate containsnucleotide molecules having a sequence which differs from the sequenceof nucleotide molecules at another distinct physical location on thesubstrate.